Methods for treating obesity and nonalcoholic fatty liver disease or nonalcoholic steatohepatitis using glucagon receptor antagonistic antibodies

ABSTRACT

The present disclosure relates to methods for treating or preventing obesity and/or nonalcoholic fatty liver diseases (NAFLDs) and/or nonalcoholic steatohepatitis (NASH) using a glucagon receptor blocking agent. In various embodiments, the present disclosure relates to methods for treating or preventing NAFLD/NASH using antigen binding and antagonizing proteins, e.g., fully human antibodies, that specifically bind to and antagonize the function of the human glucagon receptor.

RELATED PATENT APPLICATIONS

This application is a U.S. National Stage Application pursuant to 35U.S.C. § 371 of PCT/US2016/025336, filed Mar. 31, 2016, which claimsbenefit of U.S. Provisional Application No. 62/142,257, filed on Apr. 2,2015, incorporated in its entirety by reference herein.

TECHNICAL FIELD

Obesity is a complex medical disorder of appetite regulation and/ormetabolism resulting in excessive accumulation of adipose tissue mass.Obesity is an important clinical problem and is becoming an epidemicdisease in western cultures, affecting more than one-third of the USadult population. It is estimated that about 97 million adults in theUnited States are overweight or obese. Obesity is further associatedwith premature death and with a significant increase in mortality andmorbidity from stroke, myocardial infarction, congestive heart failure,coronary heart disease, and sudden death. Obesity also exacerbates manyhealth problems, both independently and in association with otherdiseases. The primary goals of obesity therapy are to reduce excess bodyweight, improve or prevent obesity-related morbidity and mortality, andmaintain long-term weight loss.

Nonalcoholic fatty liver disease (NAFLD), including its more aggressiveform nonalcoholic steatohepatitis (NASH), is also increasing in epidemicproportions concurrent with the obesity epidemic (Sowers J R et al.,Cardiorenal Med, 1:5-12, 2011). The dramatic rise in obesity and NAFLDappears to be due, in part, to consumption of a western diet (WD)containing high amounts of fat and sugar (e.g., sucrose or fructose), asfructose consumption in the US has more than doubled in the last threedecades (Barrera et al, Clin Liver Dis, 18:91-112, 2014). NAFLD ischaracterized by macrovesicular steatosis of the liver occurring inindividuals who consume little or no alcohol. The histological spectrumof NAFLD includes the presence of steatosis alone, fatty liver andinflammation. NASH is a more serious chronic liver disease characterizedby excessive fat accumulation in the liver that, for reasons that arestill incompletely understood, induces chronic inflammation which leadsto progressive fibrosis (scarring) that can lead to cirrhosis,hepatocellular carcinoma (HCC), eventual liver failure and death (Bruntet al., Am. J. Gastroenterol. 94:2467-2474, 1999; Brunt, Semin. LiverDis. 21:3-16, 2001; Takahashi Y et al., World J Gastroenterol,18:2300-2308, 2012).

Although NASH has become more and more prevalent, affecting 2%-5% ofAmericans and 2%-3% of people in the world (Neuschwander-Tetri B A, Am JMEd Sci, 330:326-335, 2005), its underlying cause is still not clear. Itmost often occurs in persons who are middle-aged and overweight orobese. Many subjects with NASH have elevated blood lipids (e.g.,cholesterol and triglycerides), hyperinsulinemia, insulin resistance,and many have diabetes or prediabetes. But not every obese person orevery subject with diabetes has NASH. Furthermore, some subjects withNASH are not obese, do not have diabetes, and have normal bloodcholesterol and lipids. NASH can occur without any apparent risk factorand can even occur in children. Thus, NASH is not simply obesity thataffects the liver. Currently, no specific therapies for NASH exist. Themost important recommendations given to persons with this disease areaerobic exercise, manipulations of diet and eating behavior, andreducing their weight (if obese or overweight).

While there are been continued advancements, there remains a pressingneed for more research on the molecular mechanisms that underlie obesityand its medical consequences, as well as new approaches for itstreatment. Similarly, there remains a pressing need for new methods oftreating or preventing NAFLDs in diabetic and non-diabetic subjects.

DISCLOSURE OF THE INVENTION

The present disclosure is based in part on the inventors' unique insightthat isolated antigen binding and antagonizing proteins thatspecifically bind to the human glucagon receptor may provide forimproved, effective therapies for treatment of diet induced obesity(DIO) and treatment of NAFLD/NASH in diabetic and non-diabetic subjects.The present inventors propose that the beneficial therapeutic effectsprovided by regulating glucose output in DIO and/or NAFLD/NASH subjects(via blocking the glucagon receptor) may include: reducing insulinresistance; reducing or preventing hyperinsulinemia, reducing orpreventing fat deposits in the liver; reducing or preventinginflammation in the liver; reducing or preventing the accumulation oflipid, e.g., hepatic triacylglycerol, hepatic diacylglycerol, andceramides; and preventing injury in the liver.

Thus, in one aspect, the present disclosure comprises a method fortreating or preventing NAFLD/NASH in a subject, comprising administeringto a subject diagnosed with NAFLD/NASH, or a subject at risk ofcontracting NAFLD/NASH, a therapeutically effective amount of anisolated antagonistic antigen binding protein that specifically binds tothe human glucagon receptor. In various embodiments, the isolatedantagonistic antigen binding protein comprises an antibody selected froma fully human antibody, a humanized antibody, a chimeric antibody, amonoclonal antibody, a polyclonal antibody, a recombinant antibody, anantigen-binding antibody fragment, a Fab, a Fab′, a Fab₂, a Fab′₂, aIgG, a IgM, a IgA, a IgE, a scFv, a dsFv, a dAb, a nanobody, a unibody,or a diabody. In various embodiments, the antibody is a fully humanmonoclonal antibody. In various embodiments, the present disclosurecomprises a method for treating NAFLD. In various embodiments, thepresent disclosure comprises a method for treating NASH.

In another aspect, the present disclosure comprises a method fortreating or preventing NAFLD/NASH in a subject, comprising (a)administering to a subject diagnosed with NAFLD/NASH, or a subject atrisk of contracting NAFLD/NASH, a therapeutically effective amount of anisolated antagonistic antigen binding protein that specifically binds tothe human glucagon receptor, and (b) an anti-obesity agent. In variousembodiments, the isolated antagonistic antigen binding protein comprisesan antibody selected from a fully human antibody, a humanized antibody,a chimeric antibody, a monoclonal antibody, a polyclonal antibody, arecombinant antibody, an antigen-binding antibody fragment, a Fab, aFab′, a Fab₂, a Fab′₂, a IgG, a IgM, a IgA, a IgE, a scFv, a dsFv, adAb, a nanobody, a unibody, or a diabody. In various embodiments, theantibody is a fully human monoclonal antibody. In various embodiments,the present disclosure comprises a method for treating NAFLD. In variousembodiments, the present disclosure comprises a method for treatingNASH. In various embodiments, the anti-obesity agent is selected fromgut-selective MTP inhibitors, CCKa agonists, 5HT2c agonists, MCR4agonists, lipase inhibitors, opioid antagonists, oleoyl-estrone,obinepitide, pramlintide (SYMLIN®), tesofensine, leptin, bromocriptine,orlistat, AOD-9604, and sibutramine.

In another aspect, the present disclosure relates to methods of treatinga subject classified as obese (e.g., having a body mass index (BMI) of30 kg/m² or more) comprising administering to the subject atherapeutically effective amount of an isolated antagonistic antigenbinding protein that specifically binds to the human glucagon receptor.In various embodiments, the isolated antagonistic antigen bindingprotein comprises an antibody selected from a fully human antibody, ahumanized antibody, a chimeric antibody, a monoclonal antibody, apolyclonal antibody, a recombinant antibody, an antigen-binding antibodyfragment, a Fab, a Fab′, a Fab₂, a Fab′₂, a IgG, a IgM, a IgA, a IgE, ascFv, a dsFv, a dAb, a nanobody, a unibody, or a diabody. In variousembodiments, the antibody is a fully human monoclonal antibody.

In another aspect, the present disclosure relates to methods of treatinga subject classified as obese (e.g., having a body mass index (BMI) of30 kg/m² or more) comprising: (a) administering to the subject atherapeutically effective amount of an isolated antagonistic antigenbinding protein that specifically binds to the human glucagon receptor,and (b) an anti-obesity agent. In various embodiments, the isolatedantagonistic antigen binding protein comprises an antibody selected froma fully human antibody, a humanized antibody, a chimeric antibody, amonoclonal antibody, a polyclonal antibody, a recombinant antibody, anantigen-binding antibody fragment, a Fab, a Fab′, a Fab₂, a Fab′₂, aIgG, a IgM, a IgA, a IgE, a scFv, a dsFv, a dAb, a nanobody, a unibody,or a diabody. In various embodiments, the antibody is a fully humanmonoclonal antibody. In various embodiments, the anti-obesity agent isselected from gut-selective MTP inhibitors, CCKa agonists, 5HT2cagonists, MCR4 agonists, lipase inhibitors, opioid antagonists,oleoyl-estrone, obinepitide, pramlintide (SYMLIN®), tesofensine, leptin,bromocriptine, orlistat, AOD-9604, and sibutramine.

In another aspect, the present disclosure relates to the use of anon-naturally occurring isolated antagonistic antigen binding proteinthat specifically binds to the human glucagon receptor for thepreparation of a medicament for treatment, prophylaxis and/or preventionof nonalcoholic steatohepatitis (NASH) in a subject in need thereof.

In another aspect, the present disclosure relates to the use of anon-naturally occurring isolated antagonistic antigen binding proteinthat specifically binds to the human glucagon receptor for thepreparation of a medicament for treatment, prophylaxis and/or preventionof nonalcoholic fatty liver disease (NAFLD) in a subject in needthereof.

In another aspect, the present disclosure relates to the use of anon-naturally occurring isolated antagonistic antigen binding proteinthat specifically binds to the human glucagon receptor for thepreparation of a medicament for treatment a subject classified as obese(e.g., having a body mass index (BMI) of 30 kg/m² or more).

In various embodiments, the isolated antagonistic binding protein bindsto a human glucagon receptor with a dissociation constant (K_(D)) of atleast about 1×10⁻⁷ M, at least about 1×10⁻⁸ M, at least about 1×10⁻⁹ M,at least about 1×10⁻¹⁰ M, at least about 1×10⁻¹¹ M, or at least about1×10⁻¹² M.

In various embodiments, the isolated antagonistic binding protein is afully human antibody which comprises the amino acid sequence encodingthe heavy chain variable region of SEQ ID NO: 2 and the amino acidsequence encoding the light chain variable region of SEQ ID NO: 3.

In various embodiments, the isolated antagonistic protein is a fullyhuman antibody which comprises the amino acid sequence encoding theheavy chain variable region of SEQ ID NO: 4 and the amino acid sequenceencoding the light chain variable region of SEQ ID NO: 5.

In various embodiments, the isolated antagonistic protein is a fullyhuman antibody which comprises the amino acid sequence encoding theheavy chain variable region of SEQ ID NO: 6 and the amino acid sequenceencoding the light chain variable region of SEQ ID NO: 7.

In various embodiments, the isolated antagonistic protein is a fullyhuman antibody which comprises a heavy chain variable region having theamino acid sequence selected from the group consisting of SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.

In various embodiments, the isolated antagonistic protein is a fullyhuman antibody which comprises a light chain variable region having theamino acid sequence selected from the group consisting of SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47.

In various embodiments, the isolated antagonistic protein is a fullyhuman antibody which comprises the amino acid sequence encoding theheavy chain variable region of SEQ ID NO: 28 and the amino acid sequenceencoding the light chain variable region of SEQ ID NO: 47.

In various embodiments, the isolated antagonistic protein is a fullyhuman antibody which comprises the amino acid sequence encoding theheavy chain of SEQ ID NO: 51 and the amino acid sequence encoding thelight chain of SEQ ID NO: 52.

In various embodiments, the isolated antagonistic antigen bindingprotein that specifically binds the human glucagon receptor will beadmixed with a pharmaceutically acceptable carrier to form apharmaceutical composition that can be systemically administered to thesubject via intravenous injection, intramuscular injection, subcutaneousinjection, intraperitoneal injection, transdermal injection,intra-arterial injection, intrasternal injection, intrathecal injection,intraventricular injection, intraurethral injection, intracranialinjection, intrasynovial injection or via infusions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a line plot depicting the in vivo effects on body weight (g)for the Vehicle group, REMD2.59 group, Pair feeding group, Normal dietgroup, and Prevention group as described herein, in a HFD DIO mousestudy, evaluated for up to twenty weeks. Treatment commenced on Day 57(i.e., start of week 9) for all groups but the Prevention group. For thePrevention group, REMD2.59C antibody was administered weekly startingfrom day 1 of HFD feeding.

FIG. 2 is a line plot depicting the in vivo effects on food consumption(kcal/g/day) for the Vehicle group, REMD2.59 group, Pair feeding group,Normal diet group, and Prevention group as described herein, in a HFDDIO mouse study, evaluated for up to twenty weeks. Treatment commencedon Day 57 (i.e., start of week 9) for all groups but the Preventiongroup. For the Prevention group, REMD2.59C antibody was administeredweekly starting from day 1 of HFD feeding.

FIG. 3 is a line plot depicting the in vivo effects on blood glucose(mmol/L) for the Vehicle group, REMD2.59 group, Pair feeding group,Normal diet group, and Prevention group as described herein, in a HFDDIO mouse study, evaluated for up to 19 weeks. Treatment commenced onDay 57 (i.e., start of week 9) for all groups but the Prevention group.For the Prevention group, REMD2.59C antibody was administered weeklystarting from day 1 of HFD feeding.

FIG. 4 is a line plot depicting the in vivo effects of repeat dosing ofREMD 2.59 on blood glucose (mmol/L) for the Vehicle group, REMD2.59group, Pair feeding group, Normal diet group, and Prevention group asdescribed herein, in a HFD DIO mouse study, evaluated at week 20.Treatment commenced on Day 57 (i.e., start of week 9) for all groups butthe Prevention group. For the Prevention group, REMD2.59C antibody wasadministered weekly starting from day 1 of HFD feeding. The oral glucosetolerance test (OGTT) was performed for all animals at the end of thestudy (i.e., week 20).

FIG. 5 is a bar graph depicting the AUC levels (mmol/L min bloodglucose) for the Vehicle group, REMD2.59 group, Pair feeding group,Normal diet group, and Prevention group as described herein, in a HFDDIO mouse study, evaluated at week 20. Treatment commenced on Day 57(i.e., start of week 9) for all groups but the Prevention group. For thePrevention group, REMD2.59C antibody was administered weekly startingfrom day 1 of HFD feeding.

FIG. 6 is a bar graph depicting the in vivo effects on triglyceride (TG)levels (mmol/L) in serum for the Vehicle group, REMD2.59 group, Pairfeeding group, Normal diet group, and Prevention group as describedherein, in a HFD DIO mouse study, evaluated for 20 weeks. Treatmentcommenced on Day 57 (i.e., start of week 9) for all groups but thePrevention group. For the Prevention group, REMD2.59C antibody wasadministered weekly starting from day 1 of HFD feeding.

FIG. 7 is a bar graph depicting the in vivo effects on total cholesterol(TCHO) levels (mmol/L) in serum for the Vehicle group, REMD2.59 group,Pair feeding group, Normal diet group, and Prevention group as describedherein, in a HFD DIO mouse study, evaluated for 20 weeks. Treatmentcommenced on Day 57 (i.e., start of week 9) for all groups but thePrevention group. For the Prevention group, REMD2.59C antibody wasadministered weekly starting from day 1 of HFD feeding.

FIG. 8 is a bar graph depicting the in vivo effects on alanineaminotransferase (ALT) levels (U/L), aspartate aminotransferase (AST)levels (U/L), gamma-glutamyl transpeptidase (GGT) levels (U/L), andalkaline phosphatase (ALP) levels (U/L) for the Vehicle group, REMD2.59group, Pair feeding group, Normal diet group, and Prevention group asdescribed herein, in a HFD DIO mouse study, evaluated at week 20.Treatment commenced on Day 57 (i.e., start of week 9) for all groups butthe Prevention group. For the Prevention group, REMD2.59C antibody wasadministered weekly starting from day 1 of HFD feeding.

FIG. 9 is a bar graph depicting the in vivo effects on triglyceride (TG)levels (mmol/L) in the liver for the Vehicle group, REMD2.59 group, Pairfeeding group, Normal diet group, and Prevention group as describedherein, in a HFD DIO mouse study, evaluated at week 20. Treatmentcommenced on Day 57 (i.e., start of week 9) for all groups but thePrevention group. For the Prevention group, REMD2.59C antibody wasadministered weekly starting from day 1 of HFD feeding.

FIG. 10 is a bar graph depicting the in vivo effects on totalcholesterol (TCHO) levels (mmol/L), high density lipoprotein cholesterol(HDL-C) levels (mg/g), and low density lipoprotein cholesterol (LDL-C)levels (mg/g) in the liver for the Vehicle group, REMD2.59 group, Pairfeeding group, Normal diet group, and Prevention group as describedherein, in a HFD DIO mouse study, evaluated at week 20. Treatmentcommenced on Day 57 (i.e., start of week 9) for all groups but thePrevention group. For the Prevention group, REMD2.59C antibody wasadministered weekly starting from day 1 of HFD feeding.

FIG. 11 is a bar graph depicting the in vivo effects on insulin levels(ng/mL) for the Vehicle group, REMD2.59 group, Pair feeding group,Normal diet group, and Prevention group as described herein, in a HFDDIO mouse study, evaluated for 20 weeks. Treatment commenced on Day 57(i.e., start of week 9) for all groups but the Prevention group. For thePrevention group, REMD2.59C antibody was administered weekly startingfrom day 1 of HFD feeding. The insulin levels on Day 57, 85, and 113were tested after 6 hours of fasting, and on Day 141 after fasting for16 hours (OGTT study was conducted on that day).

FIG. 12 is a bar graph depicting the in vivo effects on leptin levels(ng/mL) for the Vehicle group, REMD2.59 group, Pair feeding group,Normal diet group, and Prevention group as described herein, in a HFDDIO mouse study, evaluated at week 20. Treatment commenced on Day 57(i.e., start of week 9) for all groups but the Prevention group. For thePrevention group, REMD2.59C antibody was administered weekly startingfrom day 1 of HFD feeding. The insulin levels on Day 57, 85, and 113were tested after 6 hours of fasting, and on Day 141 after fasting for16 hours (OGTT study was conducted on that day).

FIG. 13 is a bar graph depicting the in vivo effects on activeglucagon-like peptide-1 (GLP-1) levels (pM) for the Vehicle group,REMD2.59 group, Pair feeding group, Normal diet group, and Preventiongroup as described herein, in a HFD DIO mouse study, evaluated at week20. Treatment commenced on Day 57 (i.e., start of week 9) for all groupsbut the Prevention group. For the Prevention group, REMD2.59C antibodywas administered weekly starting from day 1 of HFD feeding.

FIG. 14 is a bar graph depicting the wet weight (g) for white adiposetissue (WAT), liver, muscle and pancreas for the Vehicle group, REMD2.59group, Pair feeding group, Normal diet group, and Prevention group asdescribed herein, in a HFD DIO mouse study, evaluated at week 20.Treatment commenced on Day 57 (i.e., start of week 9) for all groups butthe Prevention group. For the Prevention group, REMD2.59C antibody wasadministered weekly starting from day 1 of HFD feeding.

FIG. 15 is a bar graph depicting the IHC results (% insulin area/isletarea and % glucagon are/islet area) for the Vehicle group, REMD2.59group, Pair feeding group, Normal diet group, and Prevention group asdescribed herein, in a HFD DIO mouse study, evaluated at week 20.Treatment commenced on Day 57 (i.e., start of week 9) for all groups butthe Prevention group. For the Prevention group, REMD2.59C antibody wasadministered weekly starting from day 1 of HFD feeding.

FIG. 16 depicts the results of histological H&E staining (20×10) ofvarious liver sections for the Vehicle group, REMD2.59 group, Pairfeeding group, Normal diet group, and Prevention group as describedherein, in a HFD DIO mouse study, evaluated at week 20. Treatmentcommenced on Day 57 (i.e., start of week 9) for all groups but thePrevention group. For the Prevention group, REMD2.59C antibody wasadministered weekly starting from day 1 of HFD feeding.

MODE(S) FOR CARRYING OUT THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those commonly used and well known in the art. The methodsand techniques of the present disclosure are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989) and Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates (1992), and Harlow and LaneAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1990), incorporated herein by reference.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The nomenclature used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those commonly used and well known in the art. Standardtechniques are used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofsubjects.

Definitions

The terms “peptide” “polypeptide” and “protein” each refers to amolecule comprising two or more amino acid residues joined to each otherby peptide bonds. These terms encompass, e.g., native and artificialproteins, protein fragments and polypeptide analogs (such as muteins,variants, and fusion proteins) of a protein sequence as well aspost-translationally, or otherwise covalently or non-covalently,modified proteins. A peptide, polypeptide, or protein may be monomericor polymeric. In certain embodiments, “peptides”, “polypeptides”, and“proteins” are chains of amino acids whose alpha carbons are linkedthrough peptide bonds. The terminal amino acid at one end of the chain(amino terminal) therefore has a free amino group, while the terminalamino acid at the other end of the chain (carboxy terminal) has a freecarboxyl group. As used herein, the term “amino terminus” (abbreviatedN-terminus) refers to the free α-amino group on an amino acid at theamino terminal of a peptide or to the α-amino group (imino group whenparticipating in a peptide bond) of an amino acid at any other locationwithin the peptide. Similarly, the term “carboxy terminus” refers to thefree carboxyl group on the carboxy terminus of a peptide or the carboxylgroup of an amino acid at any other location within the peptide.Peptides also include essentially any polyamino acid including, but notlimited to, peptide mimetics such as amino acids joined by an ether asopposed to an amide bond.

Polynucleotide and polypeptide sequences are indicated using standardone- or three-letter abbreviations. Unless otherwise indicated,polypeptide sequences have their amino termini at the left and theircarboxy termini at the right, and single-stranded nucleic acidsequences, and the top strand of double-stranded nucleic acid sequences,have their 5′ termini at the left and their 3′ termini at the right. Aparticular section of a polypeptide can be designated by amino acidresidue number such as amino acids 80 to 119, or by the actual residueat that site such as Ser80 to Ser119. A particular polypeptide orpolynucleotide sequence also can be described by explaining how itdiffers from a reference sequence.

Polypeptides of the disclosure include polypeptides that have beenmodified in any way and for any reason, for example, to: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (5) confer or modify other physicochemical orfunctional properties. For example, single or multiple amino acidsubstitutions (e.g., conservative amino acid substitutions) may be madein the naturally occurring sequence (e.g., in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts). A“conservative amino acid substitution” refers to the substitution in apolypeptide of an amino acid with a functionally similar amino acid. Thefollowing six groups each contain amino acids that are conservativesubstitutions for one another:

Alanine (A), Serine (S), and Threonine (T)

Aspartic acid (D) and Glutamic acid (E)

Asparagine (N) and Glutamine (Q)

Arginine (R) and Lysine (K)

Isoleucine (I), Leucine (L), Methionine (M), and Valine (V)

Phenylalanine (F), Tyrosine (Y), and Tryptophan (W)

A “non-conservative amino acid substitution” refers to the substitutionof a member of one of these classes for a member from another class. Inmaking such changes, according to certain embodiments, the hydropathicindex of amino acids may be considered. Each amino acid has beenassigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. They are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art(see, for example, Kyte et al., 1982, J. Mol. Biol. 157:105-131). It isknown that certain amino acids may be substituted for other amino acidshaving a similar hydropathic index or score and still retain a similarbiological activity. In making changes based upon the hydropathic index,in certain embodiments, the substitution of amino acids whosehydropathic indices are within ±2 is included. In certain embodiments,those that are within ±1 are included, and in certain embodiments, thosewithin ±0.5 are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, asdisclosed herein. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+−0.1);glutamate (+3.0.+−0.1); serine (+0.3); asparagine (+0.2); glutamine(+0.2); glycine (0); threonine (−0.4); proline (−0.5.+−0.1); alanine(−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine(−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3);phenylalanine (−2.5) and tryptophan (−3.4). In making changes based uponsimilar hydrophilicity values, in certain embodiments, the substitutionof amino acids whose hydrophilicity values are within ±2 is included, incertain embodiments, those that are within ±1 are included, and incertain embodiments, those within ±0.5 are included. Exemplary aminoacid substitutions are set forth in Table 1.

TABLE 1 Original Residues Exemplary Substitutions PreferredSubstitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln AspGlu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn,Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Leu Phe, Norleucine LeuNorleucine, Ile, Ile Val, Met, Ala, Phe Lys Arg, 1,4 Diamino-butyric ArgAcid, Gln, Asn Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Leu ProAla Gly Ser Thr, Ala, Cys Thr Thr Ser Trp Tyr, Phe Tyr Tyr Trp, Phe,Thr, Ser Phe Val Ile, Met, Leu, Phe, Leu Ala, Norleucine

A skilled artisan will be able to determine suitable variants ofpolypeptides as set forth herein using well-known techniques. In certainembodiments, one skilled in the art may identify suitable areas of themolecule that may be changed without destroying activity by targetingregions not believed to be important for activity. In other embodiments,the skilled artisan can identify residues and portions of the moleculesthat are conserved among similar polypeptides. In further embodiments,even areas that may be important for biological activity or forstructure may be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, the skilledartisan can predict the importance of amino acid residues in apolypeptide that correspond to amino acid residues important foractivity or structure in similar polypeptides. One skilled in the artmay opt for chemically similar amino acid substitutions for suchpredicted important amino acid residues.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of a polypeptide withrespect to its three-dimensional structure. In certain embodiments, oneskilled in the art may choose to not make radical changes to amino acidresidues predicted to be on the surface of the polypeptide, since suchresidues may be involved in important interactions with other molecules.Moreover, one skilled in the art may generate test variants containing asingle amino acid substitution at each desired amino acid residue. Thevariants can then be screened using activity assays known to thoseskilled in the art. Such variants could be used to gather informationabout suitable variants. For example, if one discovered that a change toa particular amino acid residue resulted in destroyed, undesirablyreduced, or unsuitable activity, variants with such a change can beavoided. In other words, based on information gathered from such routineexperiments, one skilled in the art can readily determine the aminoacids where further substitutions should be avoided either alone or incombination with other mutations.

The term “polypeptide fragment” and “truncated polypeptide” as usedherein refers to a polypeptide that has an amino-terminal and/orcarboxy-terminal deletion as compared to a corresponding full-lengthprotein. In certain embodiments, fragments can be, e.g., at least 5, atleast 10, at least 25, at least 50, at least 100, at least 150, at least200, at least 250, at least 300, at least 350, at least 400, at least450, at least 500, at least 600, at least 700, at least 800, at least900 or at least 1000 amino acids in length. In certain embodiments,fragments can also be, e.g., at most 1000, at most 900, at most 800, atmost 700, at most 600, at most 500, at most 450, at most 400, at most350, at most 300, at most 250, at most 200, at most 150, at most 100, atmost 50, at most 25, at most 10, or at most 5 amino acids in length. Afragment can further comprise, at either or both of its ends, one ormore additional amino acids, for example, a sequence of amino acids froma different naturally-occurring protein (e.g., an Fc or leucine zipperdomain) or an artificial amino acid sequence (e.g., an artificial linkersequence).

The terms “polypeptide variant” and “polypeptide mutant” as used hereinrefers to a polypeptide that comprises an amino acid sequence whereinone or more amino acid residues are inserted into, deleted from and/orsubstituted into the amino acid sequence relative to another polypeptidesequence. In certain embodiments, the number of amino acid residues tobe inserted, deleted, or substituted can be, e.g., at least 1, at least2, at least 3, at least 4, at least 5, at least 10, at least 25, atleast 50, at least 75, at least 100, at least 125, at least 150, atleast 175, at least 200, at least 225, at least 250, at least 275, atleast 300, at least 350, at least 400, at least 450 or at least 500amino acids in length. Variants of the present disclosure include fusionproteins.

A “derivative” of a polypeptide is a polypeptide that has beenchemically modified, e.g., conjugation to another chemical moiety suchas, for example, polyethylene glycol, albumin (e.g., human serumalbumin), phosphorylation, and glycosylation.

The term “% sequence identity” is used interchangeably herein with theterm “% identity” and refers to the level of amino acid sequenceidentity between two or more peptide sequences or the level ofnucleotide sequence identity between two or more nucleotide sequences,when aligned using a sequence alignment program. For example, as usedherein, 80% identity means the same thing as 80% sequence identitydetermined by a defined algorithm, and means that a given sequence is atleast 80% identical to another length of another sequence. In certainembodiments, the % identity is selected from, e.g., at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% or more sequence identity to agiven sequence. In certain embodiments, the % identity is in the rangeof, e.g., about 60% to about 70%, about 70% to about 80%, about 80% toabout 85%, about 85% to about 90%, about 90% to about 95%, or about 95%to about 99%.

The term “% sequence homology” is used interchangeably herein with theterm “% homology” and refers to the level of amino acid sequencehomology between two or more peptide sequences or the level ofnucleotide sequence homology between two or more nucleotide sequences,when aligned using a sequence alignment program. For example, as usedherein, 80% homology means the same thing as 80% sequence homologydetermined by a defined algorithm, and accordingly a homologue of agiven sequence has greater than 80% sequence homology over a length ofthe given sequence. In certain embodiments, the % homology is selectedfrom, e.g., at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99% ormore sequence homology to a given sequence. In certain embodiments, the% homology is in the range of, e.g., about 60% to about 70%, about 70%to about 80%, about 80% to about 85%, about 85% to about 90%, about 90%to about 95%, or about 95% to about 99%.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly available on the Internet at the NCBI website. See alsoAltschul et al., 1990, J. Mol. Biol. 215:403-10 (with special referenceto the published default setting, i.e., parameters w=4, t=17) andAltschul et al., 1997, Nucleic Acids Res., 25:3389-3402. Sequencesearches are typically carried out using the BLASTP program whenevaluating a given amino acid sequence relative to amino acid sequencesin the GenBank Protein Sequences and other public databases. The BLASTXprogram is preferred for searching nucleic acid sequences that have beentranslated in all reading frames against amino acid sequences in theGenBank Protein Sequences and other public databases. Both BLASTP andBLASTX are run using default parameters of an open gap penalty of 11.0,and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix.See id.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci.USA, 90:5873-5787 (1993)). One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is, e.g., less than about 0.1, less than about 0.01, orless than about 0.001.

The term “isolated molecule” (where the molecule is, for example, apolypeptide, a polynucleotide, or an antibody) is a molecule that byvirtue of its origin or source of derivation (1) is not associated withnaturally associated components that accompany it in its native state,(2) is substantially free of other molecules from the same species (3)is expressed by a cell from a different species, or (4) does not occurin nature. Thus, a molecule that is chemically synthesized, or expressedin a cellular system different from the cell from which it naturallyoriginates, will be “isolated” from its naturally associated components.A molecule also may be rendered substantially free of naturallyassociated components by isolation, using purification techniques wellknown in the art. Molecule purity or homogeneity may be assayed by anumber of means well known in the art. For example, the purity of apolypeptide sample may be assayed using polyacrylamide gelelectrophoresis and staining of the gel to visualize the polypeptideusing techniques well known in the art. For certain purposes, higherresolution may be provided by using HPLC or other means well known inthe art for purification.

A protein or polypeptide is “substantially pure,” “substantiallyhomogeneous,” or “substantially purified” when at least about 60% to 75%of a sample exhibits a single species of polypeptide. A substantiallypure polypeptide or protein will typically comprise about 50%, 60%, 70%,80% or 90% W/W of a protein sample, more usually about 95%, and e.g.,will be over 99% pure. Protein purity or homogeneity may be indicated bya number of means well known in the art, such as polyacrylamide gelelectrophoresis of a protein sample, followed by visualizing a singlepolypeptide band upon staining the gel with a stain well known in theart. For certain purposes, higher resolution may be provided by usingHPLC or other means well known in the art for purification.

An “antigen binding and antagonizing protein” is a protein comprising aportion that binds to an antigen and, optionally, a scaffold orframework portion that allows the antigen binding portion to adopt aconformation that promotes binding of the isolated antagonistic antigenbinding protein to the antigen. Examples of antigen binding andantagonizing proteins include antibodies, antibody fragments (e.g., anantigen binding portion of an antibody), antibody derivatives, andantibody analogs. The isolated antagonistic antigen binding protein cancomprise, for example, an alternative protein scaffold or artificialscaffold with grafted CDRs or CDR derivatives. Such scaffolds include,but are not limited to, antibody-derived scaffolds comprising mutationsintroduced to, for example, stabilize the three-dimensional structure ofthe isolated antagonistic antigen binding protein as well as whollysynthetic scaffolds comprising, for example, a biocompatible polymer.See, for example, Korndorfer et al., 2003, Proteins: Structure,Function, and Bioinformatics, Volume 53, Issue 1:121-129 (2003); Roqueet al., Biotechnol. Prog. 20:639-654 (2004). In addition, peptideantibody mimetics (“PAMs”) can be used, as well as scaffolds based onantibody mimetics utilizing fibronection components as a scaffold.

An isolated antagonistic antigen binding protein can have, for example,the structure of a naturally occurring immunoglobulin. An“immunoglobulin” is a tetrameric molecule. In a naturally occurringimmunoglobulin, each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The amino-terminal portion of eachchain includes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of each chain defines a constant region primarily responsiblefor effector function. Human light chains are classified as kappa andlambda light chains. Heavy chains are classified as mu, delta, gamma,alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG,IgA, and IgE, respectively. Within light and heavy chains, the variableand constant regions are joined by a “J” region of about 12 or moreamino acids, with the heavy chain also including a “D” region of about10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul,W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference inits entirety for all purposes). The variable regions of each light/heavychain pair form the antibody binding site such that an intactimmunoglobulin has two binding sites.

An “antibody” refers to a protein comprising one or more polypeptidessubstantially or partially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes and having specificity to a tumor antigen orspecificity to a molecule overexpressed in a pathological state. Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as subtypes ofthese genes and myriad of immunoglobulin variable region genes. Lightchains (LC) are classified as either kappa or lambda. Heavy chains (HC)are classified as gamma, mu, alpha, delta, or epsilon, which in turndefine the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively. A typical immunoglobulin (e.g., antibody) structural unitcomprises a tetramer. Each tetramer is composed of two identical pairsof polypeptide chains, each pair having one “light” (about 25 kD) andone “heavy” chain (about 50-70 kD). The N-terminus of each chain definesa variable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or V_(H)) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, C_(H1), C_(H2) and C_(H3) (and in some instances,C_(H4)). Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or V_(L)) and a light chain constant region.The light chain constant region is comprised of one domain, C_(L). TheV_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR₁, CDR₁, FR₂, CDR₂, FR₃, CDR₃, FR₄. The extent of the frameworkregion and CDRs has been defined. The sequences of the framework regionsof different light or heavy chains are relatively conserved within aspecies, such as humans. The framework region of an antibody, that isthe combined framework regions of the constituent light and heavychains, serves to position and align the CDRs in three-dimensionalspace. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM,IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG 3, IgG4, IgA1 and IgA2)or subclass.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments. Such fragments include Fab fragments, Fab′fragments, Fab₂, F(ab)′₂ fragments, single chain Fv proteins (“scFv”)and disulfide stabilized Fv proteins (“dsFv”), that bind to the targetantigen. A scFv protein is a fusion protein in which a light chainvariable region of an immunoglobulin and a heavy chain variable regionof an immunoglobulin are bound by a linker, while in dsFvs, the chainshave been mutated to introduce a disulfide bond to stabilize theassociation of the chains. While various antibody fragments are definedin terms of the digestion of an intact antibody, one of skill willappreciate that such fragments may be synthesized de novo eitherchemically or by utilizing recombinant DNA methodology. Thus, as usedherein, the term antibody encompasses e.g., monoclonal antibodies(including full-length monoclonal antibodies), polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies) formed from atleast two intact antibodies, human antibodies, humanized antibodies,camelised antibodies, chimeric antibodies, single-chain Fvs (scFv),single-chain antibodies, single domain antibodies, domain antibodies,Fab fragments, F(ab′)₂ fragments, antibody fragments that exhibit thedesired biological activity, disulfide-linked Fvs (sdFv), intrabodies,and epitope-binding fragments or antigen binding fragments of any of theabove.

A Fab fragment is a monovalent fragment having the V_(L), V_(H), C_(L)and C_(H1) domains; a F(ab′)₂ fragment is a bivalent fragment having twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment has the V_(H) and C.sub.H1 domains; an Fv fragment has theV_(L) and VdH domains of a single arm of an antibody; and a dAb fragmenthas a V_(H) domain, a V_(L) domain, or an antigen-binding fragment of aV_(H) or V_(L) domain (U.S. Pat. Nos. 6,846,634, 6,696,245, US App. Pub.No. 05/0202512, 04/0202995, 04/0038291, 04/0009507, 03/0039958, Ward etal., Nature 341:544-546 (1989)).

A single-chain antibody (scFv) is an antibody in which a V_(L) and aV_(H) region are joined via a linker (e.g., a synthetic sequence ofamino acid residues) to form a continuous protein chain wherein thelinker is long enough to allow the protein chain to fold back on itselfand form a monovalent antigen binding site (see, e.g., Bird et al.,Science 242:423-26 (1988) and Huston et al., 1988, Proc. Natl. Acad.Sci. USA 85:5879-83 (1988)). Diabodies are bivalent antibodiescomprising two polypeptide chains, wherein each polypeptide chaincomprises V_(H) and V_(L) domains joined by a linker that is too shortto allow for pairing between two domains on the same chain, thusallowing each domain to pair with a complementary domain on anotherpolypeptide chain (see, e.g., Holliger et al., 1993, Proc. Natl. Acad.Sci. USA 90:6444-48 (1993), and Poljak et al., Structure 2:1121-23(1994)). If the two polypeptide chains of a diabody are identical, thena diabody resulting from their pairing will have two identical antigenbinding sites. Polypeptide chains having different sequences can be usedto make a diabody with two different antigen binding sites. Similarly,tribodies and tetrabodies are antibodies comprising three and fourpolypeptide chains, respectively, and forming three and four antigenbinding sites, respectively, which can be the same or different.

An isolated antagonistic antigen binding protein may have one or morebinding sites. If there is more than one binding site, the binding sitesmay be identical to one another or may be different. For example, anaturally occurring human immunoglobulin typically has two identicalbinding sites, while a “bispecific” or “bifunctional” antibody has twodifferent binding sites.

The term “human antibody” includes all antibodies that have one or morevariable and constant regions derived from human immunoglobulinsequences. In one embodiment, all of the variable and constant domainsare derived from human immunoglobulin sequences (a fully humanantibody). These antibodies may be prepared in a variety of ways,examples of which are described below, including through theimmunization with an antigen of interest of a mouse that is geneticallymodified to express antibodies derived from human heavy and/or lightchain-encoding genes.

A “humanized antibody” has a sequence that differs from the sequence ofan antibody derived from a non-human species by one or more amino acidsubstitutions, deletions, and/or additions, such that the humanizedantibody is less likely to induce an immune response, and/or induces aless severe immune response, as compared to the non-human speciesantibody, when it is administered to a human subject. In one embodiment,certain amino acids in the framework and constant domains of the heavyand/or light chains of the non-human species antibody are mutated toproduce the humanized antibody. In another embodiment, the constantdomain(s) from a human antibody are fused to the variable domain(s) of anon-human species. In another embodiment, one or more amino acidresidues in one or more CDR sequences of a non-human antibody arechanged to reduce the likely immunogenicity of the non-human antibodywhen it is administered to a human subject, wherein the changed aminoacid residues either are not critical for immunospecific binding of theantibody to its antigen, or the changes to the amino acid sequence thatare made are conservative changes, such that the binding of thehumanized antibody to the antigen is not significantly worse than thebinding of the non-human antibody to the antigen. Examples of how tomake humanized antibodies may be found in U.S. Pat. Nos. 6,054,297,5,886,152 and 5,877,293.

An isolated antagonistic antigen binding protein of the presentdisclosure, including an antibody, “specifically binds” to an antigen,such as the human glucagon receptor if it binds to the antigen with ahigh binding affinity as determined by a dissociation constant (Kd, orcorresponding Kb, as defined below) value of 10⁻⁷ M or less. An isolatedantagonistic antigen binding protein that specifically binds to thehuman glucagon receptor may be able to bind to glucagon receptors fromother species as well with the same or different affinities.

An “epitope” is the portion of a molecule that is bound by an isolatedantagonistic antigen binding protein (e.g., by an antibody). An epitopecan comprise non-contiguous portions of the molecule (e.g., in apolypeptide, amino acid residues that are not contiguous in thepolypeptide's primary sequence but that, in the context of thepolypeptide's tertiary and quaternary structure, are near enough to eachother to be bound by an antigen binding and antagonizing protein).

The term “blood glucose level”, or “level of blood glucose” shall meanblood glucose concentration. In certain embodiments, a blood glucoselevel is a plasma glucose level. Plasma glucose may be determined inaccordance with, e.g., Etgen et al., Metabolism, 49(5): 684-688, 2000)or calculated from a conversion of whole blood glucose concentration inaccordance with D'Orazio et al., Clin. Chem. Lab. Med.,44(12):1486-1490, 2006.

The term “normal glucose levels” refers to mean plasma glucose values inhumans of less than about 100 mg/dL for fasting levels, and less thanabout 145 mg/dL for 2-hour post-prandial levels or 125 mg/dL for arandom glucose. The term “elevated blood glucose level” or “elevatedlevels of blood glucose” shall mean an elevated blood glucose level suchas that found in a subject demonstrating clinically inappropriate basaland postprandial hyperglycemia or such as that found in a subject inoral glucose tolerance test (oGTT), with “elevated levels of bloodglucose” being greater than about 100 mg/dL when tested under fastingconditions, and greater than about 200 mg/dL when tested at 1 hour.

The terms “glucagon inhibitor”, “glucagon suppressor” and “glucagonantagonist” are used interchangeably. Each is a molecule that detectablyinhibits glucagon signaling. The inhibition caused by an inhibitor neednot be complete so long as the inhibition is detectable using an assaythat is recognized and understood in the art as being determinative ofglucagon signaling inhibition.

A “pharmaceutical composition” refers to a composition suitable forpharmaceutical use in an animal or human. A pharmaceutical compositioncomprises a pharmacologically and/or therapeutically effective amount ofan active agent and a pharmaceutically acceptable carrier.“Pharmaceutically acceptable carrier” refers to compositions that do notproduce adverse, allergic, or other untoward reactions when administeredto an animal or a human. As used herein “pharmaceutically acceptablecarrier” refers to any of the standard pharmaceutical carriers,vehicles, buffers, and carriers, such as a phosphate buffered salinesolution, 5% aqueous solution of dextrose, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agentsand/or adjuvants. Suitable pharmaceutical carriers and formulations aredescribed in Remington's Pharmaceutical Sciences, 21st Ed. 2005, MackPublishing Co, Easton. A “pharmaceutically acceptable salt” is a saltthat can be formulated into a compound for pharmaceutical use including,e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) andsalts of ammonia or organic amines.

As used herein, a “therapeutically effective amount” of an isolatedantagonistic antigen binding protein that specifically binds the humanglucagon receptor refers to an amount of such protein that, whenprovided to a subject in accordance with the disclosed and claimedmethods effects one of the following biological activities: treatsobesity; treats NAFLD; treats NASH; or reduces, suppresses, attenuates,or inhibits one or more symptoms of NASH.

The terms “treat”, “treating” and “treatment” refer refers to anapproach for obtaining beneficial or desired clinical results. Further,references herein to “treatment” include references to curative,palliative and prophylactic treatment. For purposes of this disclosure,beneficial or desired clinical results include, but are not limited to,one or more of the following: improvement in blood glucose to withinabout 80-180 mg/dL, or to within about 80-170 mg/dL, or to within about80-160 mg/dL, or to within about 80-150 mg/dL, or to within about 80-140mg/dL, or an improvement in any one or more conditions, diseases, orsymptoms associated with, or resulting from, elevated levels of bloodglucose including, but not limited to, hyperglycemia, hyperglucanemia,and hyperinsulinemia.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise. It is understood that aspects and variations of thedisclosure described herein include “consisting” and/or “consistingessentially of” aspects and variation.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

Obesity

Obesity is a medical condition in which excess body fat has accumulatedin multiple tissues, including the liver, to the extent that it may havean adverse effect on health. Typically defined as a body mass index(BMI)(a measurement obtained by dividing a person's weight by the squareof the person's height) of 30 kg/m² or more, the prevalence of obesityhas risen significantly in the past decade in the United States and manyother developed countries and become a world-wide public health concern.

Obesity is most commonly caused by a combination of excessive foodenergy intake, lack of physical activity, and genetic susceptibility,although a few cases are caused primarily by genes, endocrine disorders,medications, or psychiatric illness. Obesity increases the likelihood ofvarious diseases, particularly heart disease, type 2 diabetes,NAFLD/NASH, obstructive sleep apnea, certain types of cancer,osteoarthritis, and asthma. Complications are either directly caused byobesity or indirectly related through mechanisms sharing a common causesuch as a poor diet or a sedentary lifestyle. The strength of the linkbetween obesity and specific conditions varies. One of the strongest isthe link with type 2 diabetes. Excess body fat underlies 64% of cases ofdiabetes in men and 77% of cases in women.

Current treatment modalities typically include diet and exerciseprograms, lifestyle management, pharmacotherapy, and surgery. Treatmentdecisions are made based on severity of obesity, seriousness ofassociated medical conditions, subject risk status, and subjectexpectations. Notable improvements in cardiovascular risk and theincidence of diabetes have been observed with weight loss of 5-10% ofbody weight, supporting clinical guidelines for the treatment of obesitythat recommend a target threshold of 10% reduction in body weight frombaseline values. Unfortunately, the available pharmacological therapiesto facilitate weight loss fail to provide adequate benefit to many obesesubjects because of side effects, contraindications or lack of positiveresponse (National Heart, Lung and Blood Institute, Clinical guidelineson the identification, evaluation, and treatment of overweight andobesity in adults: the evidence report, NIH Publication No. 98-4083,September 1998).

Bariatric surgery may be considered as a weight loss intervention forsubjects at or exceeding a BMI of 40 kg/m². Subjects with a BMI of ˜35kg/m² and with an associated serious medical condition are alsocandidates for this treatment option. Unfortunately, postoperativecomplications commonly result from bariatric surgical procedures,including bleeding, embolism or thrombosis, wound complications, deepinfections, pulmonary complications, and gastrointestinal obstruction;reoperation during the postoperative period is sometimes necessary toaddress these complications. Rates of reoperation or conversion surgerybeyond the postoperative period depend upon the type of bariatricprocedure, and in one study ranged from 17% to 31%. Intestinalabsorptive abnormalities, such as micronutrient deficiency andprotein-calorie malnutrition, also are typically seen with bypassprocedures, requiring lifelong nutrient supplementation. Major andserious adverse outcomes associated with bariatric surgery are common,observed in approximately 4 percent of procedures performed (includingdeath in 0.3 to 2 percent of all subjects receiving laparoscopic bandingor bypass surgeries, respectively).

There clearly still exists a pressing need for improved and/or newmethods of treatment of obesity, including, e.g., diet induced obesity(DIO). The present disclosure provides antigen binding and antagonizingproteins that specifically bind to the human glucagon receptor that mayprovide for improved, effective therapies for treatment of DIO indiabetic and non-diabetic subjects.

Nonalcoholic Fatty Liver Disease (NAFLD) and NonalcoholicSteatohepatitis NASH

Nonalcoholic fatty liver disease (NAFLD) is highly prevalent in theWestern population. Recent studies suggest that this disease may occurat a frequency of 70% in obese individuals and 35% in lean individuals(Wanless I R, et al., Hepatology, 12:1106-1110, 1990). NAFLD ischaracterized by macrovesicular steatosis of the liver occurring inindividuals who consume little or no alcohol. The histological spectrumof NAFLD is classified as simple steatosis alone, or nonalcoholicsteatohepatitis (NASH). Some epidemiological studies implicate dietshigher in saturated fat (Musso G, et al., Hepatology, 37:909-916, 2003).However, NAFLD is also strongly associated with the ingestion offructose, especially from sweetened beverages (Ouyang X, et al., JHepatol., 48:993-999, 2008). The classic Western diet is high in bothsaturated fats and in sugar.

Hepatic insulin resistance (which can lead to hyperglycemia and/orhyperinsulinemia) that develops with consumption of high-fat andhigh-sugar diets (e.g., simple sugar such as glucose, fructose) isclosely linked to NAFLD. Accumulating evidence suggests that hepaticinsulin resistance is caused by dysfunction in three pathways of energymetabolism (Samuel et al, Cell, 148:852-871, 2012). First, excesscarbohydrate flux (e.g., glucose, fructose) is associated withresistance to the suppressive effect of insulin on hepatic glucoseproduction and excess disposal of carbons via de novo lipogenesis (Id).Second, elevation in lipid synthesis (or reduced lipid secretion/export)leads to accumulation of hepatic triacylglycerols (TAG) which are inertbut often track with increased levels of bioactive lipid intermediatesdiacylglycerols (DAG) and ceramides that putatively lead to hepaticinsulin resistance (Kumashiro N et al, Proc Natl Acad Sci USA,108:16381-16385, 2011). Third, the hepaticsteatosis linked to insulinresistance is associated with mitochondrial dysfunction and alteredhepatic fatty acid oxidation (Rector R S et al., J Hepatol, 52:727-736,2010).

By mechanisms that are not completely understood, NAFLD may progress toa more aggressive form, nonalcoholic steatohepatitis (NASH), or progressto fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). NASH is nowaccepted as a progressive metabolic liver disease that affects 2%-5% ofAmericans and that can lead to cirrhosis and permanent liver failure(Brunt et al., supra, 1999; Brunt, supra, 2001; Ludwig et al., J.Gastroenterol. Hepatol. 12:398-403, 1997). The current model ofpathogenesis from healthy liver to NASH suggests a two-hit progression.First, insulin resistance causes lipid accumulation in hepatocytes(first hit). Secondly, it is proposed that cellular insults such asoxidative stress, lipid peroxidation, direct lipid toxicity,mitochondrial dysfunction, and/or infection causes hepatic inflammation(second hit), resulting in NASH (Brunt, supra, 2001).

The engorgement of the liver with lipid causes severe insulin resistancein the liver and abnormal glucose production (Samuel et al., J. Biol.Chem. 279:32345-32353, 2004). Steatosis caused by the insulin resistanceis believed to sensitize the liver to metabolic injury leading toinflammation, necrosis, and fibrosis (James et al., Lancet353:1634-1636, 1999; Ludwig et al., Mayo Clin. Proc. 55:434-438, 1980;Day, Gut 50:585-588, 2002; Browning et al., J. Clin. Invest.114:147-152, 2004). Thus, steatosis is a constant feature of NASH, butNASH is only distinguishable by liver biopsy. The assessment andseverity of NASH is made histologically based on the patterns anddegrees of hepatic steatosis, inflammation, and injury and, bydefinition, occurs only in the absence of significant alcoholconsumption (Brunt, supra, 2001). While steatosis is seen in both animaland human models, NASH is only fully appreciated in the human condition(Browning et al., supra, 2004). Thus, understanding the clinicalvariation observed in NASH is critical for the development oftherapeutic strategies for this condition.

Currently there are no good clinical markers that allow for theidentification of patients with NASH. Similarly, there are no therapiesto slow down or alter the course of further disease progression in NASH.Such markers and treatment for NASH are needed in the art. NASH ranks asone of the major causes of cirrhosis in America, behind hepatitis C andalcoholic liver disease. Thus, there exists a need in the art formethods of treating NASH. The present disclosure provides antigenbinding and antagonizing proteins that specifically bind to the humanglucagon receptor that may provide for improved, effective therapies fortreatment and prevention of NASH in diabetic and non-diabetic subjects.

Glucagon Receptor and Antigen Binding and Antagonizing Proteins

Glucagon is a 29 amino acid hormone processed from its pre-pro-form inthe pancreatic alpha cells by cell specific expression of prohormoneconvertase 2 (PC2), a neuroendocrine-specific protease involved in theintracellular maturation of prohormones and proneuropeptides (Furuta etal., J. Biol. Chem. 276: 27197-27202 (2001)). In vivo, glucagon is amajor counter-regulatory hormone for insulin actions. During fasting,glucagon secretion increases in response to falling glucose levels.Increased glucagon secretion stimulates glucose production by promotinghepatic glycogenolysis and gluconeogenesis (Dunning and Gerich,Endocrine Reviews, 28:253-283 (2007)). Thus glucagon counterbalances theeffects of insulin in maintaining normal levels of glucose in animals.

The biological effects of glucagon are mediated through the binding andsubsequent activation of a specific cell surface receptor, the glucagonreceptor. The glucagon receptor (GCGR) is a member of the secretinsubfamily (family B) of G-protein-coupled receptors. The human GCGR is a477 amino acid sequence GPCR and the amino acid sequence of GCGR ishighly conserved across species (Mayo et al, Pharmacological Rev.,55:167-194, (2003)). The glucagon receptor is predominantly expressed inthe liver, where it regulates hepatic glucose output, on the kidney, andon islet β-cells, reflecting its role in gluconeogenesis. The activationof the glucagon receptors in the liver stimulates the activity of adenylcyclase and phosphoinositol turnover which subsequently results inincreased expression of gluconeogenic enzymes includingphosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase(FBPase-1), and glucose-6-phosphatase (G-6-Pase). In addition, glucagonsignaling activates glycogen phosphorylase and inhibits glycogensynthase. Studies have shown that higher basal glucagon levels and lackof suppression of postprandial glucagon secretion contribute to diabeticconditions in humans (Muller et al., N Eng J Med 283: 109-115 (1970)).As such, methods of controlling and lowering blood glucose by targetingglucagon production or function using a GCGR antagonist have beenexplored.

In various embodiments, the antigen binding and antagonizing proteins ofthe present disclosure may be selected to bind to membrane-boundglucagon receptors as expressed on cells, and inhibit or block glucagonsignaling through the glucagon receptor. In various embodiments, theantigen binding and antagonizing proteins of the present disclosurespecifically bind to the human glucagon receptor. In variousembodiments, the antigen binding and antagonizing proteins binding tothe human glucagon receptor may also bind to the glucagon receptors ofother species. The polynucleotide and polypeptide sequences for severalspecies of glucagon receptor are known (see, e.g., U.S. Pat. No.7,947,809, herein incorporated by reference in its entirety for itsspecific teaching of polynucleotide and polypeptide sequences of ahuman, rat, mouse and cynomolgus glucagon receptor). In variousembodiments of the present disclosure, the antigen binding andantagonizing proteins specifically bind the human glucagon receptorhaving the amino acid sequence set forth in SEQ ID NO: 1:

Glucagon Receptor Human (Homo sapiens) amino acidsequence (Accession Number AAI04855) (SEQ ID NO: 1)MPPCQPQRPLLLLLLLLACQPQVPSAQVMDFLFEKWKLYGDQCHHNLSLLPPPTELVCNRTFDKYSCWPDTPANTTANISCPWYLPWHHKVQHRFVFKRCGPDGQWVRGPRGQPWRDASQCQMDGEEIEVQKEVAKMYSSFQVMYTVGYSLSLGALLLALAILGGLSKLHCTRNAIHANLFASFVLKASSVLVIDGLLRTRYSQKIGDDLSVSTWLSDGAVAGCRVAAVFMQYGIVANYCWLLVEGLYLHNLLGLATLPERSFFSLYLGIGWGAPMLFVVPWAVVKCLFENVQCWTSNDNMGFWWILRFPVFLAILINFFIFVRIVQLLVAKLRARQMHHTDYKFRLAKSTLTLIPLLGVHEVVFAFVTDEHAQGTLRSAKLFFDLFLSSFQGLLVAVLYCFLNKEVQSELRRRWHRWRLGKVLWEERNTSNHRASSSPGHGPPSKELQFGRGGGSQDSSAETPLAGGLPRLAESPFIn various embodiments, the antigen binding and antagonizing proteins ofthe present disclosure specifically bind glucagon receptors which haveat least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity (as calculated using methods known in the art and describedherein) to the glucagon receptors described in the cited references arealso included in the present disclosure.

The antigen binding and antagonizing proteins of the present disclosurefunction to block the interaction between glucagon and its receptor,thereby inhibiting the glucose elevating effects of glucagon. As such,the use of the antigen binding and antagonizing proteins of the presentdisclosure are an effective means of achieving normal levels of glucose,thereby ameliorating, or preventing one or more symptoms of, or longterm complications caused by diabetes including, but not limited to,hyperglycemia, hyperglucanemia, and hyperinsulinemia. The use of theantigen binding and antagonizing proteins of the present disclosure arealso an effective means of achieving normal levels of glucose innon-diabetic patients, thereby lowering the risk of hyperglycemia,hyperglucanemia, and hyperinsulinemia in subjects having disordersincluding, but not limited to, obesity or NAFLDs, and for treating suchnon-diabetic disorders.

Methods of generating antibodies that bind to antigens such as the humanglucagon receptor are known to those skilled in the art. For example, amethod for generating a monoclonal antibody that binds specifically to atargeted antigen polypeptide may comprise administering to a mouse anamount of an immunogenic composition comprising the targeted antigenpolypeptide effective to stimulate a detectable immune response,obtaining antibody-producing cells (e.g., cells from the spleen) fromthe mouse and fusing the antibody-producing cells with myeloma cells toobtain antibody-producing hybridomas, and testing the antibody-producinghybridomas to identify a hybridoma that produces a monocolonal antibodythat binds specifically to the targeted antigen polypeptide. Onceobtained, a hybridoma can be propagated in a cell culture, optionally inculture conditions where the hybridoma-derived cells produce themonoclonal antibody that binds specifically to targeted antigenpolypeptide. The monoclonal antibody may be purified from the cellculture. A variety of different techniques are then available fortesting an antigen/antibody interaction to identify particularlydesirable antibodies.

Other suitable methods of producing or isolating antibodies of therequisite specificity can used, including, for example, methods whichselect recombinant antibody from a library, or which rely uponimmunization of transgenic animals (e.g., mice) capable of producing afull repertoire of human antibodies. See e.g., Jakobovits et al., Proc.Natl. Acad. Sci. (U.S.A.), 90: 2551-2555, 1993; Jakobovits et al.,Nature, 362: 255-258, 1993; Lonberg et al., U.S. Pat. No. 5,545,806; andSurani et al., U.S. Pat. No. 5,545,807.

Antibodies can be engineered in numerous ways. They can be made assingle-chain antibodies (including small modular immunopharmaceuticalsor SMIPs™), Fab and F(ab′)₂ fragments, etc. Antibodies can be humanized,chimerized, deimmunized, or fully human. Numerous publications set forththe many types of antibodies and the methods of engineering suchantibodies. For example, see U.S. Pat. Nos. 6,355,245; 6,180,370;5,693,762; 6,407,213; 6,548,640; 5,565,332; 5,225,539; 6,103,889; and5,260,203.

Chimeric antibodies can be produced by recombinant DNA techniques knownin the art. For example, a gene encoding the Fc constant region of amurine (or other species) monoclonal antibody molecule is digested withrestriction enzymes to remove the region encoding the murine Fc, and theequivalent portion of a gene encoding a human Fc constant region issubstituted (see Robinson et al., International Patent PublicationPCT/US86/02269; Akira, et al., European Patent Application 184,187;Taniguchi, M., European Patent Application 171,496; Morrison et al.,European Patent Application 173,494; Neuberger et al., InternationalApplication WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabillyet al., European Patent Application 125,023; Better et al., Science,240:1041-1043, 1988; Liu et al., Proc. Natl. Acad. Sci. (U.S.A.),84:3439-3443, 1987; Liu et al., J. Immunol., 139:3521-3526, 1987; Sun etal., Proc. Natl. Acad. Sci. (U.S.A.), 84:214-218, 1987; Nishimura etal., Canc. Res., 47:999-1005, 1987; Wood et al., Nature, 314:446-449,1985; and Shaw et al., J. Natl Cancer Inst., 80:1553-1559, 1988).

Methods for humanizing antibodies have been described in the art. Insome embodiments, a humanized antibody has one or more amino acidresidues introduced from a source that is nonhuman, in addition to thenonhuman CDRs. Humanization can be essentially performed following themethod of Winter and co-workers (Jones et al., Nature, 321:522-525,1986; Riechmann et al., Nature, 332:323-327, 1988; Verhoeyen et al.,Science, 239:1534-1536, 1988), by substituting hypervariable regionsequences for the corresponding sequences of a human antibody.Accordingly, such “humanized” antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567) wherein substantially less than an intact humanvariable region has been substituted by the corresponding sequence froma nonhuman species. In practice, humanized antibodies are typicallyhuman antibodies in which some hypervariable region residues andpossibly some framework region residues are substituted by residues fromanalogous sites in rodent antibodies.

U.S. Pat. No. 5,693,761 to Queen et al, discloses a refinement on Winteret al. for humanizing antibodies, and is based on the premise thatascribes avidity loss to problems in the structural motifs in thehumanized framework which, because of steric or other chemicalincompatibility, interfere with the folding of the CDRs into thebinding-capable conformation found in the mouse antibody. To addressthis problem, Queen teaches using human framework sequences closelyhomologous in linear peptide sequence to framework sequences of themouse antibody to be humanized. Accordingly, the methods of Queen focuson comparing framework sequences between species. Typically, allavailable human variable region sequences are compared to a particularmouse sequence and the percentage identity between correspondentframework residues is calculated. The human variable region with thehighest percentage is selected to provide the framework sequences forthe humanizing project. Queen also teaches that it is important toretain in the humanized framework, certain amino acid residues from themouse framework critical for supporting the CDRs in a binding-capableconformation. Potential criticality is assessed from molecular models.Candidate residues for retention are typically those adjacent in linearsequence to a CDR or physically within 6 Å of any CDR residue.

In other approaches, the importance of particular framework amino acidresidues is determined experimentally once a low-avidity humanizedconstruct is obtained, by reversion of single residues to the mousesequence and assaying antigen binding as described by Riechmann et al,1988. Another example approach for identifying important amino acids inframework sequences is disclosed by U.S. Pat. No. 5,821,337 to Carter etal, and by U.S. Pat. No. 5,859,205 to Adair et al. These referencesdisclose specific Kabat residue positions in the framework, which, in ahumanized antibody may require substitution with the correspondent mouseamino acid to preserve avidity.

Another method of humanizing antibodies, referred to as “frameworkshuffling”, relies on generating a combinatorial library with nonhumanCDR variable regions fused in frame into a pool of individual humangermline frameworks (Dall'Acqua et al., Methods, 36:43, 2005). Thelibraries are then screened to identify clones that encode humanizedantibodies which retain good binding.

The choice of human variable regions, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable region of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence that is closest to that of the rodent is then accepted as thehuman framework region (framework region) for the humanized antibody(Sims et al., J. Immunol., 151:2296, 1993; Chothia et al., J. Mol.Biol., 196:901, 1987). Another method uses a particular framework regionderived from the consensus sequence of all human antibodies of aparticular subgroup of light or heavy chain variable regions. The sameframework may be used for several different humanized antibodies (Carteret al., Proc. Natl. Acad. Sci. (U.S.A.), 89:4285, 1992; Presta et al.,J. Immunol., 151:2623, 1993).

The choice of nonhuman residues to substitute into the human variableregion can be influenced by a variety of factors. These factors include,for example, the rarity of the amino acid in a particular position, theprobability of interaction with either the CDRs or the antigen, and theprobability of participating in the interface between the light andheavy chain variable domain interface. (See, for example, U.S. Pat. Nos.5,693,761, 6,632,927, and 6,639,055). One method to analyze thesefactors is through the use of three-dimensional models of the nonhumanand humanized sequences. Three-dimensional immunoglobulin models arecommonly available and are familiar to those skilled in the art.Computer programs are available that illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, e.g., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, nonhuman residues can be selected and substituted for humanvariable region residues in order to achieve the desired antibodycharacteristic, such as increased affinity for the target antigen(s).

Methods for making fully human antibodies have been described in theart. By way of example, a method for producing an anti-GCGR antibody orantigen binding antibody fragment thereof comprises the steps ofsynthesizing a library of human antibodies on phage, screening thelibrary with GCGR or an antibody binding portion thereof, isolatingphage that bind GCGR, and obtaining the antibody from the phage. By wayof another example, one method for preparing the library of antibodiesfor use in phage display techniques comprises the steps of immunizing anon-human animal comprising human immunoglobulin loci with GCGR or anantigenic portion thereof to create an immune response, extractingantibody-producing cells from the immunized animal; isolating RNAencoding heavy and light chains of antibodies of the disclosure from theextracted cells, reverse transcribing the RNA to produce cDNA,amplifying the cDNA using primers, and inserting the cDNA into a phagedisplay vector such that antibodies are expressed on the phage.Recombinant anti-GCGR antibodies of the disclosure may be obtained inthis way.

Again, by way of example, recombinant human anti-GCGR antibodies of thedisclosure can also be isolated by screening a recombinant combinatorialantibody library. Preferably the library is a scFv phage displaylibrary, generated using human V_(L) and V_(H) cDNAs prepared from mRNAisolated from B cells. Methods for preparing and screening suchlibraries are known in the art. Kits for generating phage displaylibraries are commercially available (e.g., the Pharmacia RecombinantPhage Antibody System, catalog no. 27-9400-01; and the StratageneSurfZAP™ phage display kit, catalog no. 240612). There also are othermethods and reagents that can be used in generating and screeningantibody display libraries (see, e.g., U.S. Pat. No. 5,223,409; PCTPublication Nos. WO 92/18619, WO 91/17271, WO 92/20791, WO 92/15679, WO93/01288, WO 92/01047, WO 92/09690; Fuchs et al., Bio/Technology,9:1370-1372 (1991); Hay et al., Hum. Antibod. Hybridomas, 3:81-85, 1992;Huse et al., Science, 246:1275-1281, 1989; McCafferty et al., Nature,348:552-554, 1990; Griffiths et al., EMBO J., 12:725-734, 1993; Hawkinset al., J. Mol. Biol., 226:889-896, 1992; Clackson et al., Nature,352:624-628, 1991; Gram et al., Proc. Natl. Acad. Sci. (U.S.A.),89:3576-3580, 1992; Garrad et al., Bio/Technology, 9:1373-1377, 1991;Hoogenboom et al., Nuc. Acid Res., 19:4133-4137, 1991; and Barbas etal., Proc. Natl. Acad. Sci. (U.S.A.), 88:7978-7982, 1991), allincorporated herein by reference.

Human antibodies are also produced by immunizing a non-human, transgenicanimal comprising within its genome some or all of human immunoglobulinheavy chain and light chain loci with a human IgE antigen, e.g., aXenoMouse™ animal (Abgenix, Inc./Amgen, Inc.—Fremont, Calif.).XenoMouse™ mice are engineered mouse strains that comprise largefragments of human immunoglobulin heavy chain and light chain loci andare deficient in mouse antibody production. See, e.g., Green et al.,Nature Genetics, 7:13-21, 1994 and U.S. Pat. Nos. 5,916,771, 5,939,598,5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, 6,130,364,6,162,963 and 6,150,584. XenoMouse™ mice produce an adult-like humanrepertoire of fully human antibodies and generate antigen-specific humanantibodies. In some embodiments, the XenoMouse™ mice containapproximately 80% of the human antibody V gene repertoire throughintroduction of megabase sized, germline configuration fragments of thehuman heavy chain loci and kappa light chain loci in yeast artificialchromosome (YAC). In other embodiments, XenoMouse™ mice further containapproximately all of the human lambda light chain locus. See Mendez etal., Nature Genetics, 15:146-156, 1997; Green and Jakobovits, J. Exp.Med., 188:483-495, 1998; and WO 98/24893.

In various embodiments, the isolated antagonistic antigen bindingprotein of the present disclosure utilize an antibody or antigen bindingantibody fragment thereof is a polyclonal antibody, a monoclonalantibody or antigen-binding fragment thereof, a recombinant antibody, adiabody, a chimerized or chimeric antibody or antigen-binding fragmentthereof, a humanized antibody or antigen-binding fragment thereof, afully human antibody or antigen-binding fragment thereof, a CDR-graftedantibody or antigen-binding fragment thereof, a single chain antibody,an Fv, an Fd, an Fab, an Fab′, or an F(ab′)₂, and synthetic orsemi-synthetic antibodies.

In various embodiments, the isolated antagonistic antigen bindingprotein of the present disclosure utilize an antibody or antigen-bindingfragment that binds to a glucagon receptor antigen with a dissociationconstant (K_(D)) of, e.g., at least about 1×10⁻⁷ M, at least about1×10⁻⁸ M, at least about 1×10⁻⁹ M, at least about 1×10⁻¹⁰M, at leastabout 1×10⁻¹¹ M, or at least about 1×10⁻¹² M. In various embodiments,the isolated antagonistic antigen binding protein of the presentdisclosure utilize an antibody or antigen-binding fragment that binds toa glucagon receptor antigen with a dissociation constant (K_(D)) in therange of, e.g., at least about 1×10⁻⁷ M to at least about 1×10⁻⁸ M, atleast about 1×10⁻⁸ M to at least about 1×10⁻⁹ M, at least about 1×10⁻⁹ Mto at least about 1×10⁻¹⁰M, at least about 1×10⁻¹⁰ M to at least about1×10⁻¹¹ M, or at least about 1×10⁻¹¹ M to at least about 1×10⁻¹² M.

Antibodies to the glucagon receptor have been described in, e.g., U.S.Pat. Nos. 5,770,445; 7,947,809; 7,968,686; 8,545,847; 8,771,696;9,102,732; 9,248,189; European patent application EP2074149A2; EP patentEP0658200B1; U.S. patent publications 2009/0041784; 2009/0252727;2013/0344538; 2014/0335091; and 20160075778 and PCT publicationWO2008/036341. In various embodiments of the present invention, theisolated antagonistic antigen binding protein is an anti-GCGR(“antagonistic”) antibody or antigen-binding fragment which comprisesthe polynucleotide and polypeptide sequences set forth in, e.g., U.S.Pat. Nos. 7,947,809, and 8,158,759, each herein incorporated byreference in its entirety for its specific teaching of polynucleotideand polypeptide sequences of various anti-GCGR antibodies orantigen-binding fragments.

In various embodiments of the present disclosure the antibody may be ananti-GCGR antibody that has the same or higher antigen-binding affinityas that of the antibody comprising the heavy chain variable regionsequence as set forth in SEQ ID NO: 2. In various embodiments, theantibody may be an anti-GCGR antibody which binds to the same epitope asthe antibody comprising the heavy chain variable region sequence as setforth in SEQ ID NO: 2. In various embodiments, the antibody is ananti-GCGR antibody which competes with the antibody comprising the heavychain variable region sequence as set forth in SEQ ID NO: 2. In variousembodiments, the antibody may be an anti-GCGR antibody which comprisesat least one (such as two or three) CDRs of the heavy chain variableregion sequence as set forth in SEQ ID NO: 2. In various embodiments,the antibody may be an anti-GCGR antibody which comprises the heavychain variable region sequence as set forth in SEQ ID NO: 2. In variousembodiments, the antibody may be an anti-GCGR antibody which comprisesthe heavy chain variable region sequence as set forth in SEQ ID NO: 2:

(SEQ ID NO: 2) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTTVTVSS

In various embodiments of the present disclosure the antibody may be ananti-GCGR antibody that has the same or higher antigen-binding affinityas that of the antibody comprising the light chain variable regionsequence as set forth in SEQ ID NO: 3. In various embodiments, theantibody may be an anti-GCGR antibody which binds to the same epitope asthe antibody comprising the light chain variable region sequence as setforth in SEQ ID NO: 3. In various embodiments, the antibody is ananti-GCGR antibody which competes with the antibody comprising the lightchain variable region sequence as set forth in SEQ ID NO: 3. In variousembodiments, the antibody may be an anti-GCGR antibody which comprisesat least one (such as two or three) CDRs of the light chain variableregion sequence as set forth in SEQ ID NO: 3. In various embodiments,the antibody may be an anti-GCGR antibody which comprises the lightchain variable region sequence as set forth in SEQ ID NO: 3. In variousembodiments, the antibody may be an anti-GCGR antibody which comprisesthe light chain variable region sequence as set forth in SEQ ID NO: 3:

(SEQ ID NO: 3) DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGG GTKVEIK

In various embodiments, the antibody contains an amino acid sequencethat shares an observed homology of, e.g., at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% with the sequences of SEQ IDNOS: 2 or 3.

In various embodiments of the present disclosure the antibody may be ananti-GCGR antibody that has the same or higher antigen-binding affinityas that of the antibody comprising the heavy chain variable regionsequence as set forth in SEQ ID NO: 4. In various embodiments, theantibody may be an anti-GCGR antibody which binds to the same epitope asthe antibody comprising the heavy chain variable region sequence as setforth in SEQ ID NO: 4. In various embodiments, the antibody is ananti-GCGR antibody which competes with the antibody comprising the heavychain variable region sequence as set forth in SEQ ID NO: 4. In variousembodiments, the antibody may be an anti-GCGR antibody which comprisesat least one (such as two or three) CDRs of the heavy chain variableregion sequence as set forth in SEQ ID NO: 4. In various embodiments,the antibody may be an anti-GCGR antibody which comprises the heavychain variable region sequence as set forth in SEQ ID NO: 4. In variousembodiments, the antibody may be an anti-GCGR antibody which comprisesthe heavy chain variable region sequence as set forth in SEQ ID NO: 4:

(SEQ ID NO: 4) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTTVTVSS

In various embodiments of the present disclosure the antibody may be ananti-GCGR antibody that has the same or higher antigen-binding affinityas that of the antibody comprising the light chain variable regionsequence as set forth in SEQ ID NO: 5. In various embodiments, theantibody may be an anti-GCGR antibody which binds to the same epitope asthe antibody comprising the light chain variable region sequence as setforth in SEQ ID NO: 5. In various embodiments, the antibody is ananti-GCGR antibody which competes with the antibody comprising the lightchain variable region sequence as set forth in SEQ ID NO: 5. In variousembodiments, the antibody may be an anti-GCGR antibody which comprisesat least one (such as two or three) CDRs of the light chain variableregion sequence as set forth in SEQ ID NO: 5. In various embodiments,the antibody may be an anti-GCGR antibody which comprises the lightchain variable region sequence as set forth in SEQ ID NO: 5. In variousembodiments, the antibody may be an anti-GCGR antibody which comprisesthe light chain variable region sequence as set forth in SEQ ID NO: 5:

(SEQ ID NO: 5) DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFVTYYCLQHNSNPLTFGG GTKVEIK

In various embodiments, the antibody contains an amino acid sequencethat shares an observed homology of, e.g., at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% with the sequences of SEQ IDNOS: 4 or 5.

In various embodiments of the present disclosure the antibody may be ananti-GCGR antibody that has the same or higher antigen-binding affinityas that of the antibody comprising the heavy chain variable regionsequence as set forth in SEQ ID NO: 6. In various embodiments, theantibody may be an anti-GCGR antibody which binds to the same epitope asthe antibody comprising the heavy chain variable region sequence as setforth in SEQ ID NO: 6. In various embodiments, the antibody is ananti-GCGR antibody which competes with the antibody comprising the heavychain variable region sequence as set forth in SEQ ID NO: 6. In variousembodiments, the antibody may be an anti-GCGR antibody which comprisesat least one (such as two or three) CDRs of the heavy chain variableregion sequence as set forth in SEQ ID NO: 6. In various embodiments,the antibody may be an anti-GCGR antibody which comprises the heavychain variable region sequence as set forth in SEQ ID NO: 6. In variousembodiments, the antibody may be an anti-GCGR antibody which comprisesthe heavy chain variable region sequence as set forth in SEQ ID NO: 6:

(SEQ ID NO: 6) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTTVTVSS

In various embodiments of the present disclosure the antibody may be ananti-GCGR antibody that has the same or higher antigen-binding affinityas that of the antibody comprising the light chain variable regionsequence as set forth in SEQ ID NO: 7. In various embodiments, theantibody may be an anti-GCGR antibody which binds to the same epitope asthe antibody comprising the light chain variable region sequence as setforth in SEQ ID NO: 7. In various embodiments, the antibody is ananti-GCGR antibody which competes with the antibody comprising the lightchain variable region sequence as set forth in SEQ ID NO: 7. In variousembodiments, the antibody may be an anti-GCGR antibody which comprisesat least one (such as two or three) CDRs of the light chain variableregion sequence as set forth in SEQ ID NO: 7. In various embodiments,the antibody may be an anti-GCGR antibody which comprises the lightchain variable region sequence as set forth in SEQ ID NO: 7. In variousembodiments, the antibody may be an anti-GCGR antibody which comprisesthe light chain variable region sequence as set forth in SEQ ID NO: 7:

(SEQ ID NO: 7) DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLESGVPSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGT KVEIK

In various embodiments, the antibody contains an amino acid sequencethat shares an observed homology of, e.g., at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% with the sequences of SEQ IDNOS: 6 or 7.

In various embodiments of the present disclosure the antibody may be ananti-GCGR antibody that has the same or higher antigen-binding affinityas that of the chimeric antibody comprising the heavy chain sequence asset forth in SEQ ID NO: 8. In various embodiments, the antibody may bean anti-GCGR antibody which binds to the same epitope as the antibodycomprising the heavy chain sequence as set forth in SEQ ID NO: 8. Invarious embodiments, the antibody is an anti-GCGR antibody whichcompetes with the antibody comprising the heavy chain sequence as setforth in SEQ ID NO: 8. In various embodiments, the antibody may be ananti-GCGR antibody which comprises at least one (such as two or three)CDRs of the heavy chain sequence as set forth in SEQ ID NO: 8. Invarious embodiments, the antibody may be an anti-GCGR antibody whichcomprises the heavy chain sequence as set forth in SEQ ID NO: 8. Invarious embodiments, the antibody may be an anti-GCGR antibody whichcomprises the heavy chain sequence as set forth in SEQ ID NO: 8:

(SEQ ID NO: 8) MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVIWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCS VLHEGLHNHHTEKSLSHSPGK

In various embodiments of the present disclosure the antibody may be ananti-GCGR antibody that has the same or higher antigen-binding affinityas that of the chimeric antibody comprising the light chain sequence asset forth in SEQ ID NO: 9. In various embodiments, the antibody may bean anti-GCGR antibody which binds to the same epitope as the antibodycomprising the light chain sequence as set forth in SEQ ID NO: 9. Invarious embodiments, the antibody is an anti-GCGR antibody whichcompetes with the antibody comprising the light chain sequence as setforth in SEQ ID NO: 9. In various embodiments, the antibody may be ananti-GCGR antibody which comprises at least one (such as two or three)CDRs of the light chain sequence as set forth in SEQ ID NO: 9. Invarious embodiments, the antibody may be an anti-GCGR antibody whichcomprises the light chain sequence as set forth in SEQ ID NO: 9. Invarious embodiments, the antibody may be an anti-GCGR antibody whichcomprises the light chain sequence as set forth in SEQ ID NO: 9:

(SEQ ID NO: 9) MDMRVPAQLLGLLLLWFPGARCDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYOQKPGKAPKRLIYAASSLESGVPSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

In various embodiments, the antibody contains an amino acid sequencethat shares an observed homology of, e.g., at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% with the sequences of SEQ IDNOS: 8 or 9.

In various embodiments of the present disclosure the antibody may be ananti-GCGR antibody which comprises a heavy chain variable regionsequence selected from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ IDNO: 27, and SEQ ID NO: 28, and a light chain variable region sequenceselected from, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ IDNO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46,and SEQ ID NO: 47. In various embodiments, the antibody contains anamino acid sequence that shares an observed homology of, e.g., at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% with the sequences ofSEQ ID NOS: 10-28 or SEQ ID NOS: 29-47.

Examples of Anti-GCGR Antibodies

HCVR LCVR SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO:6 SEQ ID NO: 7 SEQ ID NO: 10 SEQ ID NO: 29 SEQ ID NO: 11 SEQ ID NO: 30SEQ ID NO: 12 SEQ ID NO: 31 SEQ ID NO: 13 SEQ ID NO: 32 SEQ ID NO: 14SEQ ID NO: 33 SEQ ID NO: 15 SEQ ID NO: 34 SEQ ID NO: 16 SEQ ID NO: 35SEQ ID NO: 17 SEQ ID NO: 36 SEQ ID NO: 18 SEQ ID NO: 37 SEQ ID NO: 19SEQ ID NO: 38 SEQ ID NO: 20 SEQ ID NO: 39 SEQ ID NO: 21 SEQ ID NO: 40SEQ ID NO: 22 SEQ ID NO: 41 SEQ ID NO: 23 SEQ ID NO: 42 SEQ ID NO: 24SEQ ID NO: 43 SEQ ID NO: 25 SEQ ID NO: 44 SEQ ID NO: 26 SEQ ID NO: 45SEQ ID NO: 27 SEQ ID NO: 46 SEQ ID NO: 28 SEQ ID NO: 47

In various embodiments, the isolated antagonistic antibody is a fullyhuman antibody which comprises the amino acid sequence encoding theheavy chain variable region of SEQ ID NO: 28 and the amino acid sequenceencoding the light chain variable region of SEQ ID NO: 47.

An isolated anti-GCGR antibody, antibody fragment, or antibodyderivative of the present disclosure can comprise any constant regionknown in the art. The light chain constant region can be, for example, akappa- or lambda-type light chain constant region, e.g., a human kappa-or lambda-type light chain constant region. The heavy chain constantregion can be, for example, an alpha-, delta-, epsilon-, gamma-, ormu-type heavy chain constant regions, e.g., a human alpha-, delta-,epsilon-, gamma-, or mu-type heavy chain constant region. In variousembodiments, the light or heavy chain constant region is a fragment,derivative, variant, or mutein of a naturally occurring constant region.

Techniques are known for deriving an antibody of a different subclass orisotype from an antibody of interest, i.e., subclass switching. Thus,IgG antibodies may be derived from an IgM antibody, for example, andvice versa. Such techniques allow the preparation of new antibodies thatpossess the antigen-binding properties of a given antibody (the parentantibody), but also exhibit biological properties associated with anantibody isotype or subclass different from that of the parent antibody.Recombinant DNA techniques may be employed. Cloned DNA encodingparticular antibody polypeptides may be employed in such procedures,e.g., DNA encoding the constant domain of an antibody of the desiredisotype. See also Lanitto et al., Methods Mol. Biol. 178:303-16 (2002).

In various embodiments, an isolated antigen binding protein of thepresent disclosure comprises the constant light chain kappa region asset forth in SEQ ID NO: 48, or a fragment thereof. In variousembodiments, an isolated antigen binding protein of the presentdisclosure comprises the constant light chain lambda region as set forthin SEQ ID NO: 49, or a fragment thereof. In various embodiments, anisolated antigen binding protein of the present disclosure comprises aIgG2 heavy chain constant region set forth in SEQ ID NO: 50, or afragment thereof.

In various embodiments, an isolated antagonistic antigen binding proteinof the present disclosure is a fully human anti-GCGR antibody thatcomprises a heavy chain sequence as set forth in SEQ ID NO: 51 and alight chain as set forth in SEQ ID NO: 52.

In various embodiments of the present disclosure, the isolatedantagonistic antigen binding protein is a hemibody. A “hemibody” is animmunologically-functional immunoglobulin construct comprising acomplete heavy chain, a complete light chain and a second heavy chain Fcregion paired with the Fc region of the complete heavy chain. A linkercan, but need not, be employed to join the heavy chain Fc region and thesecond heavy chain Fc region. In various embodiments, the hemibody is amonovalent antigen binding protein comprising (i) an intact light chain,and (ii) a heavy chain fused to an Fc region (e.g., an IgG2 Fc region).Methods for preparing hemibodies are described in, e.g., U.S. patentapplication 2012/0195879, herein incorporated by reference in itsentirety herein for purposes of teaching the preparation of suchhemibodies.

Pharmaceutical Compositions

In another aspect, the present disclosure provides a pharmaceuticalcomposition comprising an isolated antagonistic antigen binding proteinas described herein, with one or more pharmaceutically acceptablecarrier(s). The pharmaceutical compositions and methods of usesdescribed herein also encompass embodiments of combinations(co-administration) with other active agents, as detailed below.

Generally, the antagonistic antigen binding proteins of the presentdisclosure are suitable to be administered as a formulation inassociation with one or more pharmaceutically acceptable carrier(s). Theterm ‘carrier’ is used herein to describe any ingredient other than thecompound(s) of the disclosure. The choice of carrier(s) will to a largeextent depend on factors such as the particular mode of administration,the effect of the carrier on solubility and stability, and the nature ofthe dosage form. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike that are physiologically compatible. Some examples ofpharmaceutically acceptable carriers are water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, the composition will includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Additional examples ofpharmaceutically acceptable substances are wetting agents or minoramounts of auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody. Pharmaceutical compositions of the present disclosureand methods for their preparation will be readily apparent to thoseskilled in the art. Such compositions and methods for their preparationmay be found, for example, in Remington's Pharmaceutical Sciences, 19thEdition (Mack Publishing Company, 1995). The pharmaceutical compositionsare generally formulated as sterile, substantially isotonic and in fullcompliance with all GMP regulations of the U.S. Food and DrugAdministration.

The pharmaceutical compositions of the present disclosure are typicallysuitable for parenteral administration. As used herein, “parenteraladministration” of a pharmaceutical composition includes any route ofadministration characterized by physical breaching of a tissue of asubject and administration of the pharmaceutical composition through thebreach in the tissue, thus generally resulting in the directadministration into the blood stream, into muscle, or into an internalorgan. Parenteral administration thus includes, but is not limited to,administration of a pharmaceutical composition by injection of thecomposition, by application of the composition through a surgicalincision, by application of the composition through a tissue-penetratingnon-surgical wound, and the like. In particular, parenteraladministration is contemplated to include, but is not limited to,subcutaneous injection, intraperitoneal injection, intramuscularinjection, intrasternal injection, intravenous injection, intraarterialinjection, intrathecal injection, intraventricular injection,intraurethral injection, intracranial injection, intrasynovial injectionor infusions; or kidney dialytic infusion techniques.

A pharmaceutical composition of the present disclosure can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present disclosure. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded. Numerous reusable pen and autoinjector deliverydevices have applications in the subcutaneous delivery of apharmaceutical composition of the present disclosure including, but notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™(Disetronic Medical Systems, Burghdorf, Switzerland), and HUMALOG MIX75/25™, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co.,Indianapolis, Ind.).

Formulations of a pharmaceutical composition suitable for parenteraladministration typically generally comprise the active ingredientcombined with a pharmaceutically acceptable carrier, such as sterilewater or sterile isotonic saline. Such formulations may be prepared,packaged, or sold in a form suitable for bolus administration or forcontinuous administration. Injectable formulations may be prepared,packaged, or sold in unit dosage form, such as in ampoules or inmulti-dose containers containing a preservative. Formulations forparenteral administration include, but are not limited to, suspensions,solutions, emulsions in oily or aqueous vehicles, pastes, and the like.Such formulations may further comprise one or more additionalingredients including, but not limited to, suspending, stabilizing, ordispersing agents. In one embodiment of a formulation for parenteraladministration, the active ingredient is provided in dry (i.e. powder orgranular) form for reconstitution with a suitable vehicle (e.g. sterilepyrogen-free water) prior to parenteral administration of thereconstituted composition. Parenteral formulations also include aqueoussolutions which may contain carriers such as salts, carbohydrates andbuffering agents (preferably to a pH of from 3 to 9), but, for someapplications, they may be more suitably formulated as a sterilenon-aqueous solution or as a dried form to be used in conjunction with asuitable vehicle such as sterile, pyrogen-free water. Exemplaryparenteral administration forms include solutions or suspensions insterile aqueous solutions, for example, aqueous propylene glycol ordextrose solutions. Such dosage forms can be suitably buffered, ifdesired. Other parentally-administrable formulations which are usefulinclude those which comprise the active ingredient in microcrystallineform, or in a liposomal preparation. Formulations for parenteraladministration may be formulated to be immediate and/or modifiedrelease. Modified release formulations include delayed-, sustained-,pulsed-, controlled-, targeted and programmed release.

For example, in one aspect, sterile injectable solutions can be preparedby incorporating the isolated antagonistic antigen binding protein inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, methods of preparation such as vacuum drying andfreeze-drying yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin. In various embodiments, the injectable compositions will beadministered using commercially available disposable injectable devices.

The isolated antagonistic antigen binding protein of the presentdisclosure can be administered intranasally or by inhalation, typicallyin the form of a dry powder (either alone, as a mixture, or as a mixedcomponent particle, for example, mixed with a suitable pharmaceuticallyacceptable carrier) from a dry powder inhaler, as an aerosol spray froma pressurized container, pump, spray, atomiser (preferably an atomiserusing electrohydrodynamics to produce a fine mist), or nebulizer, withor without the use of a suitable propellant, or as nasal drops.

The pressurized container, pump, spray, atomizer, or nebulizer generallycontains a solution or suspension of an isolated antagonistic antigenbinding protein of the disclosure comprising, for example, a suitableagent for dispersing, solubilizing, or extending release of the active,a propellant(s) as solvent.

Prior to use in a dry powder or suspension formulation, the drug productis generally micronized to a size suitable for delivery by inhalation(typically less than 5 microns). This may be achieved by any appropriatecomminuting method, such as spiral jet milling, fluid bed jet milling,supercritical fluid processing to form nanoparticles, high pressurehomogenization, or spray drying.

Capsules, blisters and cartridges for use in an inhaler or insufflatormay be formulated to contain a powder mix of the isolated antagonisticantigen binding protein of the disclosure, a suitable powder base and aperformance modifier.

Suitable flavours, such as menthol and levomenthol, or sweeteners, suchas saccharin or saccharin sodium, may be added to those formulations ofthe disclosure intended for inhaled/intranasal administration.

Formulations for inhaled/intranasal administration may be formulated tobe immediate and/or modified release. Modified release formulationsinclude delayed-, sustained-, pulsed-, controlled-, targeted andprogrammed release.

In the case of dry powder inhalers and aerosols, the dosage unit isdetermined by means of a valve which delivers a metered amount. Units inaccordance with the disclosure are typically arranged to administer ametered dose or “puff” of an antibody of the disclosure. The overalldaily dose will typically be administered in a single dose or, moreusually, as divided doses throughout the day.

The isolated antagonistic antigen binding protein of the presentdisclosure may also be formulated for an oral administration. Oraladministration may involve swallowing, so that the compound enters thegastrointestinal tract, and/or buccal, lingual, or sublingualadministration by which the compound enters the blood stream directlyfrom the mouth. Formulations suitable for oral administration includesolid, semi-solid and liquid systems such as tablets; soft or hardcapsules containing multi- or nano-particulates, liquids, or powders;lozenges (including liquid-filled); chews; gels; fast dispersing dosageforms; films; ovules; sprays; and buccal/mucoadhesive patches.

Pharmaceutical compositions intended for oral use may be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents selected from the group consisting of sweetening agents inorder to provide a pharmaceutically elegant and palatable preparation.For example, to prepare orally deliverable tablets, the isolatedantagonistic antigen binding protein is mixed with at least onepharmaceutical carrier, and the solid formulation is compressed to forma tablet according to known methods, for delivery to thegastrointestinal tract. The tablet composition is typically formulatedwith additives, e.g. a saccharide or cellulose carrier, a binder such asstarch paste or methyl cellulose, a filler, a disintegrator, or otheradditives typically usually used in the manufacture of medicalpreparations. To prepare orally deliverable capsules, DHEA is mixed withat least one pharmaceutical carrier, and the solid formulation is placedin a capsular container suitable for delivery to the gastrointestinaltract. Compositions comprising isolated antagonistic antigen bindingprotein may be prepared as described generally in Remington'sPharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa.18042) at Chapter 89, which is herein incorporated by reference.

In various embodiments, the pharmaceutical compositions are formulatedas orally deliverable tablets containing isolated antagonistic antigenbinding protein in admixture with non-toxic pharmaceutically acceptablecarriers which are suitable for manufacture of tablets. These carriersmay be inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example, maize starch, gelatin or acacia, andlubricating agents, for example, magnesium stearate, stearic acid, ortalc. The tablets may be uncoated or they may be coated with knowntechniques to delay disintegration and absorption in thegastrointestinal track and thereby provide a sustained action over alonger period of time. For example, a time delay material such asglyceryl monostearate or glyceryl distearate alone or with a wax may beemployed.

In various embodiments, the pharmaceutical compositions are formulatedas hard gelatin capsules wherein the isolated antagonistic antigenbinding protein is mixed with an inert solid diluent, for example,calcium carbonate, calcium phosphate, or kaolin or as soft gelatincapsules wherein the isolated antagonistic antigen binding protein ismixed with an aqueous or an oil medium, for example, arachis oil, peanutoil, liquid paraffin or olive oil.

Liquid formulations include suspensions, solutions, syrups and elixirs.Such formulations may be employed as fillers in soft or hard capsules(made, for example, from gelatin or hydroxypropylmethylcellulose) andtypically comprise a carrier, for example, water, ethanol, polyethyleneglycol, propylene glycol, methylcellulose, or a suitable oil, and one ormore emulsifying agents and/or suspending agents. Liquid formulationsmay also be prepared by the reconstitution of a solid, for example, froma sachet.

Any method for administering peptides, proteins or antibodies acceptedin the art may suitably be employed for administering the isolatedantagonistic antigen binding protein of the disclosure.

Methods of Treatment

Due to their interaction with the glucagon receptor, the present antigenbinding and antagonizing proteins are useful for lowering blood glucoselevels by regulating gluconeogenesis and glycogenlysis and also for thetreatment of a wide range of conditions and disorders in which blockingthe interaction of glucagon with its receptor is beneficial, while alsoreducing and or eliminating one or more of the unwanted side effectsassociated with the current treatments.

In one aspect of the present disclosure, a method for treating a subjectdiagnosed with a disorder or condition characterized by excessive levelsof glucagon (hypergluconemia) and/or blood glucose comprisingadministering to the subject a therapeutically effective amount of anisolated antagonistic antigen binding protein that specifically binds tothe human glucagon receptor, is provided. In various embodiments, theantigen binding and antagonizing proteins are fully human monoclonalantibodies and the disorder is obesity. In various embodiments, theantigen binding and antagonizing proteins are fully human monoclonalantibodies and the disorder is NAFLD. In various embodiments, theantigen binding and antagonizing proteins are fully human monoclonalantibodies and the disorder is NASH.

An antagonistic antigen binding protein, in particular a human antibodyaccording to the present disclosure, need not effect a complete cure, oreradicate every symptom or manifestation of a disease, to constitute aviable therapeutic agent. As is recognized in the pertinent field, drugsemployed as therapeutic agents may reduce the severity of a givendisease state, but need not abolish every manifestation of the diseaseto be regarded as useful therapeutic agents. Similarly, aprophylactically administered treatment need not be completely effectivein preventing the onset of a condition in order to constitute a viableprophylactic agent. Simply reducing the impact of a disease (forexample, by reducing the number or severity of its symptoms, or byincreasing the effectiveness of another treatment, or by producinganother beneficial effect), or reducing the likelihood that the diseasewill occur or worsen in a subject, is sufficient. One embodiment of thedisclosure is directed to a method comprising administering to a subjectan isolated antagonistic antigen binding protein such as a humanantibody in an amount and for a time sufficient to induce a sustainedimprovement over baseline of an indicator that reflects the severity ofthe particular disorder.

In various embodiments of the present disclosure, obesity is defined asBMI of 30 kg/m² or more (National Institute of Health, ClinicalGuidelines on the Identification, Evaluation, and Treatment ofOverweight and Obesity in Adults (1998)). In various other embodiments,the present disclosure is also intended to include a disease, disorder,or condition that is characterized by a body mass index (BMI) of 25kg/m² or more, 26 kg/m² or more, 27 kg/m² or more, 28 kg/m² or more, 29kg/m² or more, 29.5 kg/m² or more, or 29.9 kg/m² or more, all of whichare typically referred to as overweight.

An isolated antagonistic antigen binding protein that specifically bindsthe human glucagon receptor, in particular, the fully human antibodiesof the disclosure, may be administered, e.g., once or more than once, atregular intervals over a period of time. In various embodiments, a fullyhuman antibody is administered over a period of at least once a month ormore, e.g., for one, two, or three months or even indefinitely. Fortreating chronic conditions, long-term treatment is generally mosteffective. However, for treating acute conditions, administration forshorter periods, e.g. from one to six weeks, may be sufficient. Ingeneral, the fully human antibody is administered until the subjectmanifests a medically relevant degree of improvement over baseline forthe chosen indicator or indicators.

One example of therapeutic regimens provided herein comprisesubcutaneous injection of an isolated antagonistic antigen bindingprotein once a week, or once every two weeks, at an appropriate dosage,to treat a condition in which blood glucose levels play a role. Weeklyor monthly administration of isolated antagonistic antigen bindingprotein would be continued until a desired result is achieved, e.g., thesubject's symptoms subside. Treatment may resume as needed, or,alternatively, maintenance doses may be administered.

A subject's levels of blood glucose may be monitored before, duringand/or after treatment with an isolated antagonistic antigen bindingprotein such as a human antibody, to detect changes, if any, in theirlevels. For some disorders, the incidence of elevated blood glucose mayvary according to such factors as the stage of the disease. Knowntechniques may be employed for measuring glucose levels. Glucagon levelsmay also be measured in the subject's blood using known techniques, forexample, ELISA.

A therapeutically effective dose can be estimated initially from cellculture assays by determining an IC₅₀. A dose can then be formulated inanimal models to achieve a circulating plasma concentration range thatincludes the IC₅₀ as determined in cell culture. Such information can beused to more accurately determine useful doses in humans. Levels inplasma may be measured by, e.g., HPLC or immunoassays using theanti-idiotypic antibodies specific to the therapeutic drug. The exactcomposition, route of administration and dosage can be chosen by theindividual physician in view of the subject's condition.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus can be administered, several divided doses (multiple or repeat ormaintenance) can be administered over time and the dose can beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the present disclosure will be dictatedprimarily by the unique characteristics of the antibody and theparticular therapeutic or prophylactic effect to be achieved.

Thus, the skilled artisan would appreciate, based upon the disclosureprovided herein, that the dose and dosing regimen is adjusted inaccordance with methods well-known in the therapeutic arts. That is, themaximum tolerable dose can be readily established, and the effectiveamount providing a detectable therapeutic benefit to a subject may alsobe determined, as can the temporal requirements for administering eachagent to provide a detectable therapeutic benefit to the subject.Accordingly, while certain dose and administration regimens areexemplified herein, these examples in no way limit the dose andadministration regimen that may be provided to a subject in practicingthe present disclosure.

It is to be noted that dosage values may vary with the type and severityof the condition to be ameliorated, and may include single or multipledoses. It is to be further understood that for any particular subject,specific dosage regimens should be adjusted over time according to theindividual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.Further, the dosage regimen with the compositions of this disclosure maybe based on a variety of factors, including the type of disease, theage, weight, sex, medical condition of the subject, the severity of thecondition, the route of administration, and the particular antibodyemployed. Thus, the dosage regimen can vary widely, but can bedetermined routinely using standard methods. For example, doses may beadjusted based on pharmacokinetic or pharmacodynamic parameters, whichmay include clinical effects such as toxic effects and/or laboratoryvalues. Thus, the present disclosure encompasses intra-subjectdose-escalation as determined by the skilled artisan. Determiningappropriate dosages and regimens are well-known in the relevant art andwould be understood to be encompassed by the skilled artisan onceprovided the teachings disclosed herein.

For administration to human patients, the total monthly dose of theisolated antagonistic antigen binding protein of the disclosure can bein the range of 0.5-1200 mg per patient, 0.5-1100 mg per patient,0.5-1000 mg per patient, 0.5-900 mg per patient, 0.5-800 mg per patient,0.5-700 mg per patient, 0.5-600 mg per patient, 0.5-500 mg per patient,0.5-400 mg per patient, 0.5-300 mg per patient, 0.5-200 mg per patient,0.5-100 mg per patient, 0.5-50 mg per patient, 1-1200 mg per patient,1-1100 mg per patient, 1-1000 mg per patient, 1-900 mg per patient,1-800 mg per patient, 1-700 mg per patient, 1-600 mg per patient, 1-500mg per patient, 1-400 mg per patient, 1-300 mg per patient, 1-200 mg perpatient, 1-100 mg per patient, or 1-50 mg per patient depending, ofcourse, on the mode of administration. For example, an intravenousmonthly dose can require about 1-1000 mg/patient. In variousembodiments, the isolated antagonistic antigen binding protein of thedisclosure can be administered at an intravenous monthly dose of about1-500 mg per patient. In various embodiments, the isolated antagonisticantigen binding protein of the disclosure can be administered at anintravenous monthly dose of about 1-400 mg per patient. In variousembodiments, the isolated antagonistic antigen binding protein of thedisclosure can be administered at an intravenous monthly dose of about1-300 mg per patient. In various embodiments, the isolated antagonisticantigen binding protein of the disclosure can be administered at anintravenous monthly dose of about 1-200 mg per patient. In variousembodiments, the isolated antagonistic antigen binding protein of thedisclosure can be administered, at an intravenous monthly dose of about1-150 mg per patient. In various embodiments, the isolated antagonisticantigen binding protein of the disclosure can be administered or at anintravenous monthly dose of about 1-100 mg/patient. In variousembodiments, the isolated antagonistic antigen binding protein of thedisclosure can be administered at an intravenous monthly dose of about1-50 mg per patient. The total monthly dose can be administered insingle or divided doses and can, at the physician's discretion, falloutside of the typical ranges given herein.

An exemplary, non-limiting weekly, or bi-weekly dosing range for atherapeutically or prophylactically effective amount of an isolatedantagonistic antigen binding protein of the disclosure can be 0.001 to100 mg/kg body weight, 0.001 to 90 mg/kg, 0.001 to 80 mg/kg, 0.001 to 70mg/kg, 0.001 to 60 mg/kg, 0.001 to 50 mg/kg, 0.001 to 40 mg/kg, 0.001 to30 mg/kg, 0.001 to 20 mg/kg, 0.001 to 10 mg/kg, 0.001 to 5 mg/kg, 0.001to 4 mg/kg, 0.001 to 3 mg/kg, 0.001 to 2 mg/kg, 0.001 to 1 mg/kg, 0.010to 50 mg/kg, 0.010 to 40 mg/kg, 0.010 to 30 mg/kg, 0.010 to 20 mg/kg,0.010 to 10 mg/kg, 0.010 to 5 mg/kg, 0.010 to 4 mg/kg, 0.010 to 3 mg/kg,0.010 to 2 mg/kg, 0.010 to 1 mg/kg, 0.1 to 50 mg/kg, 0.1 to 40 mg/kg,0.1 to 30 mg/kg, 0.1 to 20 mg/kg, 0.1 to 10 mg/kg, 0.1 to 5 mg/kg, 0.1to 4 mg/kg, 0.1 to 3 mg/kg, 0.1 to 2 mg/kg, 0.1 to 1 mg/kg, 1 to 50mg/kg, 1 to 40 mg/kg, 1 to 30 mg/kg, 1 to 25 mg/kg, 1 to 20 mg/kg, 1 to15 mg/kg, 1 to 10 mg/kg, 1 to 7.5 mg/kg, 1 to 5 mg/kg, 1 to 4 mg/kg, 1to 3 mg/kg, 1 to 2 mg/kg, or 1 mg/kg body weight. It is to be noted thatdosage values may vary with the type and severity of the condition to bealleviated. It is to be further understood that for any particularpatient, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

In various embodiments, the total dose administered will achieve aplasma antibody concentration in the range of, e.g., about 1 to 1000μg/ml, about 1 to 750 μg/ml, about 1 to 500 μg/ml, about 1 to 250 μg/ml,about 10 to 1000 μg/ml, about 10 to 750 μg/ml, about 10 to 500 μg/ml,about 10 to 250 μg/ml, about 20 to 1000 μg/ml, about 20 to 750 μg/ml,about 20 to 500 μg/ml, about 20 to 250 μg/ml, about 30 to 1000 μg/ml,about 30 to 750 μg/ml, about 30 to 500 μg/ml, about 30 to 250 μg/ml.

In various embodiments, either as monotherapy, or in combination with ananti-obesity agent, the weekly or bi-weekly dose for a therapeuticallyeffective amount of an isolated antagonistic antigen binding protein ofthe disclosure will be 0.01 mg/kg body weight. In various embodiments,the weekly or bi-weekly dose for a therapeutically effective amount ofan isolated antagonistic antigen binding protein of the disclosure willbe 0.025 mg/kg body weight. In various embodiments, the weekly orbi-weekly dose for a therapeutically effective amount of an isolatedantagonistic antigen binding protein of the disclosure will be 0.05mg/kg body weight. In various embodiments, the weekly or bi-weekly dosefor a therapeutically effective amount of an isolated antagonisticantigen binding protein of the disclosure will be 0.075 mg/kg bodyweight. In various embodiments, the weekly or bi-weekly dose for atherapeutically effective amount of an isolated antagonistic antigenbinding protein of the disclosure will be 0.1 mg/kg body weight. Invarious embodiments, the weekly or bi-weekly dose for a therapeuticallyeffective amount of an isolated antagonistic antigen binding protein ofthe disclosure will be 0.25 mg/kg body weight. In various embodiments,the weekly or bi-weekly dose for a therapeutically effective amount ofan isolated antagonistic antigen binding protein of the disclosure willbe 0.5 mg/kg body weight. In various embodiments, the weekly orbi-weekly dose for a therapeutically effective amount of an isolatedantagonistic antigen binding protein of the disclosure will be 0.75mg/kg body weight. In various embodiments, the weekly or bi-weekly dosefor a therapeutically effective amount of an isolated antagonisticantigen binding protein of the disclosure will be 1 mg/kg body weight.In various embodiments, the weekly or bi-weekly dose for atherapeutically effective amount of an isolated antagonistic antigenbinding protein of the disclosure will be 1.5 mg/kg body weight. Invarious embodiments, the weekly or bi-weekly dose for a therapeuticallyeffective amount of an isolated antagonistic antigen binding protein ofthe disclosure will be 2 mg/kg body weight. In various embodiments, theweekly or bi-weekly dose for a therapeutically effective amount of anisolated antagonistic antigen binding protein of the disclosure will be2.5 mg/kg body weight. In various embodiments, the weekly or bi-weeklydose for a therapeutically effective amount of an isolated antagonisticantigen binding protein of the disclosure will be 3 mg/kg body weight.In various embodiments, the weekly or bi-weekly dose for atherapeutically effective amount of an isolated antagonistic antigenbinding protein of the disclosure will be 3.5 mg/kg body weight. Invarious embodiments, the weekly or bi-weekly dose for a therapeuticallyeffective amount of an isolated antagonistic antigen binding protein ofthe disclosure will be 4 mg/kg body weight. In various embodiments, theweekly or bi-weekly dose for a therapeutically effective amount of anisolated antagonistic antigen binding protein of the disclosure will be4.5 mg/kg body weight. In various embodiments, the weekly or bi-weeklydose for a therapeutically effective amount of an isolated antagonisticantigen binding protein of the disclosure will be 5 mg/kg body weight.In various embodiments, the weekly or bi-weekly dose for atherapeutically effective amount of an isolated antagonistic antigenbinding protein of the disclosure will be 5.5 mg/kg body weight. Invarious embodiments, the weekly or bi-weekly dose for a therapeuticallyeffective amount of an isolated antagonistic antigen binding protein ofthe disclosure will be 6 mg/kg body weight. In various embodiments, theweekly or bi-weekly dose for a therapeutically effective amount of anisolated antagonistic antigen binding protein of the disclosure will be6.5 mg/kg body weight. In various embodiments, the weekly or bi-weeklydose for a therapeutically effective amount of an isolated antagonisticantigen binding protein of the disclosure will be 7 mg/kg body weight.In various embodiments, the weekly or bi-weekly dose for atherapeutically effective amount of an isolated antagonistic antigenbinding protein of the disclosure will be 7.5 mg/kg body weight. Invarious embodiments, the weekly or bi-weekly dose for a therapeuticallyeffective amount of an isolated antagonistic antigen binding protein ofthe disclosure will be 8 mg/kg body weight. In various embodiments, theweekly or bi-weekly dose for a therapeutically effective amount of anisolated antagonistic antigen binding protein of the disclosure will be8.5 mg/kg body weight. In various embodiments, the weekly or bi-weeklydose for a therapeutically effective amount of an isolated antagonisticantigen binding protein of the disclosure will be 9 mg/kg body weight.In various embodiments, the weekly or bi-weekly dose for atherapeutically effective amount of an isolated antagonistic antigenbinding protein of the disclosure will be 9.5 mg/kg body weight. Invarious embodiments, the weekly or bi-weekly dose for a therapeuticallyeffective amount of an isolated antagonistic antigen binding protein ofthe disclosure will be 10 mg/kg body weight.

Toxicity and therapeutic index of the pharmaceutical compositions of thedisclosure can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effective dose is the therapeutic indexand it can be expressed as the ratio LD₅₀/ED₅₀. Compositions thatexhibit large therapeutic indices are generally preferred.

In various embodiments, single or multiple administrations of thepharmaceutical compositions are administered depending on the dosage andfrequency as required and tolerated by the subject. In any event, thecomposition should provide a sufficient quantity of at least one of theisolated antagonistic antigen binding protein disclosed herein toeffectively treat the subject. The dosage can be administered once butmay be applied periodically until either a therapeutic result isachieved or until side effects warrant discontinuation of therapy.

The dosing frequency of the administration of the isolated antagonisticantigen binding protein pharmaceutical composition depends on the natureof the therapy and the particular disease being treated. The subject canbe treated at regular intervals, such as weekly or monthly, until adesired therapeutic result is achieved. Exemplary dosing frequenciesinclude, but are not limited to: once weekly without break; once weekly,every other week; once every 2 weeks; once every 3 weeks; weakly withoutbreak for 2 weeks, then monthly; weakly without break for 3 weeks, thenmonthly; monthly; once every other month; once every three months; onceevery four months; once every five months; or once every six months, oryearly.

As used herein, the terms “co-administration”, “co-administered” and “incombination with”, referring to the isolated antagonistic antigenbinding protein of the present disclosure and one or more othertherapeutic agent(s), is intended to mean, and does refer to and includethe following: simultaneous administration of such combination ofisolated antagonistic antigen binding protein of the disclosure andtherapeutic agent(s) to a subject in need of treatment, when suchcomponents are formulated together into a single dosage form whichreleases said components at substantially the same time to said subject;substantially simultaneous administration of such combination ofisolated antagonistic antigen binding protein of the disclosure andtherapeutic agent(s) to a subject in need of treatment, when suchcomponents are formulated apart from each other into separate dosageforms which are taken at substantially the same time by said subject,whereupon said components are released at substantially the same time tosaid subject; sequential administration of such combination of isolatedantagonistic antigen binding protein of the disclosure and therapeuticagent(s) to a subject in need of treatment, when such components areformulated apart from each other into separate dosage forms which aretaken at consecutive times by said subject with a significant timeinterval between each administration, whereupon said components arereleased at substantially different times to said subject; andsequential administration of such combination of isolated antagonisticantigen binding protein of the disclosure and therapeutic agent(s) to asubject in need of treatment, when such components are formulatedtogether into a single dosage form which releases said components in acontrolled manner whereupon they are concurrently, consecutively, and/oroverlappingly released at the same and/or different times to saidsubject, where each part may be administered by either the same or adifferent route.

Suitable pharmaceutical agents that may be used in combination with thecompounds of the present invention include anti-obesity agents(including appetite suppressants), anti-diabetic agents,anti-hyperglycemic agents, lipid lowering agents, and anti-hypertensiveagents.

Suitable anti-obesity agents (some of which may also act asanti-diabetic agents as well) include 11β-hydroxy steroiddehydrogenase-1 (11β-HSD type 1) inhibitors, stearoyl-CoA desaturase-1(SCD-1) inhibitor, MCR-4 agonists, cholecystokinin-A (CCK-A) agonists,monoamine reuptake inhibitors (such as sibutramine), sympathomimeticagents, β₃ adrenergic agonists, dopamine agonists (such asbromocriptine), melanocyte-stimulating hormone analogs, 5HT2c agonists,melanin concentrating hormone antagonists, leptin (the OB protein),leptin analogs, leptin agonists, galanin antagonists, lipase inhibitors(such as tetrahydrolipstatin, i.e. orlistat), anorectic agents (such asa bombesin agonist), neuropeptide-Y antagonists (e.g., NPY Y5antagonists such as velneperit), PYY₃₋₃₆ (including analogs thereof),BRS3 modulator, mixed antagonists of opiod receptor subtypes,thyromimetic agents, dehydroepiandrosterone or an analog thereof,glucocorticoid agonists or antagonists, orexin antagonists,glucagon-like peptide-1 agonists, ciliary neurotrophic factors (such asAXOKINE™ available from Regeneron Pharmaceuticals, Inc., Tarrytown, N.Y.and Procter & Gamble Company, Cincinnati, Ohio), human agouti-relatedprotein (AGRP) inhibitors, histamine 3 antagonists or inverse agonists,neuromedin U agonists, MTP/ApoB inhibitors (e.g., gut-selective MTPinhibitors, such as dirlotapide, JTT130, Usistapide, SLx4090), opioidantagonist, mu opioid receptor modulators, including but not limited toGSK1521498, MetAp2 inhibitors, including but not limited to ZGN-433,agents with mixed modulatory activity at 2 or more of glucagon, GIP andGLP1 receptors, such as MAR-701 or ZP2929, norepinephrine transporterinhibitors, cannabinoid-1-receptor antagonist/inverse agonists, ghrelinagonists/antagonists, oxyntomodulin and analogs, monoamine uptakeinhibitors, such as but not limited to tesofensine, an orexinantagonist, combination agents (such as bupropion plus zonisamide,pramlintide plus metreleptin, bupropion plus naltrexone, phentermineplus topiramate), and the like.

In various embodiments, the anti-obesity agent is selected fromgut-selective MTP inhibitors (e.g., dirlotapide, mitratapide andimplitapide, R56918 (CAS No. 403987) and CAS No. 913541-47-6), CCKaagonists (e.g.,N-benzyl-2-[4-(1H-indol-3-ylmethyl)-5-oxo-1-phenyl-4,5-dihydro-2,3,6,10b—tetraaza-benzo[e]azulen-6-yl]-N-isopropyl-acetamide(described in PCT Publication No. WO 2005/116034 or US Publication No.2005-0267100 A1), 5HT2c agonists (e.g., lorcaserin), MCR4 agonist (e.g.,compounds described in U.S. Pat. No. 6,818,658), lipase inhibitor (e.g.,Cetilistat), PYY₃₋₃₆ (as used herein “PYY₃₋₃₆” includes analogs, such aspeglated PYY₃₋₃₆ e.g., those described in US Publication 2006/0178501),opioid antagonists (e.g., naltrexone), oleoyl-estrone (CAS No.180003-17-2), obinepitide (TM30338), pramlintide (SYMLIN™), tesofensine(NS2330), leptin, bromocriptine, orlistat, AOD-9604 (CAS No.221231-10-3) and sibutramine.

In various embodiments, the combination therapy comprises administeringthe isolated antagonistic antigen binding protein composition and thesecond agent composition simultaneously, either in the samepharmaceutical composition or in separate pharmaceutical compositions.In various embodiments, isolated antagonistic antigen binding proteincomposition and the second agent composition are administeredsequentially, i.e., the isolated antagonistic antigen binding proteincomposition is administered either prior to or after the administrationof the second agent composition.

In various embodiments, the administrations of the isolated antagonisticantigen binding protein composition and the second agent composition areconcurrent, i.e., the administration period of the isolated antagonisticantigen binding protein composition and the second agent compositionoverlap with each other.

In various embodiments, the administrations of the isolated antagonisticantigen binding protein composition and the second agent composition arenon-concurrent. For example, in various embodiments, the administrationof the isolated antagonistic antigen binding protein composition isterminated before the second agent composition is administered. Invarious embodiments, the administration second agent composition isterminated before the isolated antagonistic antigen binding proteincomposition is administered.

In various embodiments, the present disclosure comprises a method fortreating an overweight or obese subject comprising administering to thesubject a therapeutically effective amount of an isolated antagonisticantigen binding protein that specifically binds to the human glucagonreceptor.

In various embodiments, the present disclosure comprises a method fortreating an overweight or obese subject comprising administering to thesubject: (a) a therapeutically effective amount of an isolatedantagonistic antigen binding protein that specifically binds to thehuman glucagon receptor; and (b) an anti-obesity agent.

In various embodiments, the present disclosure comprises a method fortreating or preventing NAFLD/NASH in a subject, comprising administeringto a subject diagnosed with NAFLD/NASH, or a subject at risk ofcontracting NAFLD/NASH, a therapeutically effective amount of anisolated antagonistic antigen binding protein that specifically binds tothe human glucagon receptor. In various embodiments, the antibody is afully human monoclonal antibody. In various embodiments, the presentdisclosure comprises a method for treating NAFLD. In variousembodiments, the present disclosure comprises a method for treatingNASH.

In various embodiments, the present disclosure comprises a method fortreating or preventing NAFLD/NASH in a subject, comprising administeringto a subject diagnosed with NAFLD/NASH, or a subject at risk ofcontracting NAFLD/NASH, (a) a therapeutically effective amount of anisolated antagonistic antigen binding protein that specifically binds tothe human glucagon receptor; and (b) an anti-obesity agent. In variousembodiments, the antibody is a fully human monoclonal antibody. Invarious embodiments, the present disclosure comprises a method fortreating NAFLD. In various embodiments, the present disclosure comprisesa method for treating NASH.

In another aspect, the present disclosure provides methods for treatinga subject who is at risk of developing NASH (e.g., subjects who areoverweight or obese or subjects with NAFLD) comprising administering tothe subject a therapeutically effective amount of an isolatedantagonistic antigen binding protein that specifically binds to thehuman glucagon receptor.

In another aspect, the present disclosure relates to the use of anon-naturally occurring isolated antagonistic antigen binding proteinthat specifically binds to the human glucagon receptor for thepreparation of a medicament for treatment, prophylaxis and/or preventionof nonalcoholic steatohepatitis (NASH) in a subject in need thereof.

In another aspect, the present disclosure relates to the use of anon-naturally occurring isolated antagonistic antigen binding proteinthat specifically binds to the human glucagon receptor for thepreparation of a medicament for treatment, prophylaxis and/or preventionof nonalcoholic fatty liver disease (NAFLD) in a subject in needthereof.

In another aspect, the present disclosure relates to the use of anon-naturally occurring isolated antagonistic antigen binding proteinthat specifically binds to the human glucagon receptor for thepreparation of a medicament for treatment a subject classified as obese(e.g., having a body mass index (BMI) of 30 kg/m² or more).

The invention having been described, the following examples are offeredby way of illustration, and not limitation.

Example 1

In this example, the relationship between regulating glucose output andthe development of obesity in various DIO murine models is evaluated.Specifically, the in vivo activity of an anti-GCGR antibody whichcomprises the heavy chain sequence set forth in SEQ ID NO: 8 and thelight chain sequence set forth in SEQ ID NO: 9 (“REMD2.59C”) isevaluated in a 20 week DIO murine model using wild-type C57BL/6 mice.Wild-type C57BL/6 mice are commonly used for obesity research, becausethey show increasing body fat mass, hyperglycemia, and hyperinsulinemiawhen they are fed a high fat diet (“HFD”)(REbuffe-Scrive, M et al.,Metabolism, 42:1405-1409, 1993; Surwit, R S., Metabolism 44:645-651,1995).

In this study, three groups of 10 each wild-type C57BL/6J mice (male,age 4-6 weeks, 20-22 g) are fed ad libitum with a high fat diet (HFD)for 8 weeks (hereinafter “Vehicle Group” or “REMD2.59 Group” or “PairFeeding Group”). One group of 10 wild-type C57BL/6J mice (male, age 4-6weeks, 20-22 g) are fed ad libitum with normal diet (“chow”) for 8 weeks(hereinafter “Normal Diet Group”). One group of 8 wild-type C57BL/6J(male, age 4-6 weeks, 20-22 g) are fed with a HFD and dosed weekly with7.5 mg/kg REMD2.59 antibody for 8 weeks (starting on day 1) (hereinafter“Prevention Group”). The mice are kept in laminar flow rooms at constanttemperature and humidity with one animal in each cage. Animals arehoused in polycarbonate cages and in an environmentally monitored,well-ventilated room maintained at a temperature of (22±2° C.) and arelative humidity of 40%-70%. Fluorescent lighting provided illuminationapproximately 12 hours per day. The bedding material is soft wood, whichis changed once per week. All procedures were conducted in accordancewith the National Institutes of Health (NIH) guidelines for the care anduse of laboratory animals.

At 8 weeks following the start of the HFD diet, the “Vehicle Group” andthe “Pair Feeding Group” remain on HFD and the “Normal Diet Group”remains on chow and are all dosed weekly (starting on day 57 and up thruweek 20) via subcutaneous injection with vehicle (PBS). The HFD“REMD2.59 Group” is dosed weekly (starting on day 57 and up thru week20) via subcutaneous injection with 7.5 mg/kg (10 mL/kg) REMD2.59Cantibody. The HFD “Prevention Group” continues to be dosed weekly upthru week 20 via subcutaneous injection with 7.5 mg/kg (10 mL/kg)REMD2.59C antibody. The Study group assignments are outlined in Table 2below.

TABLE 2 Weekly Diet Treatment Treatment Treatment Group (Week-1-20)Dose, mg/kg, Start Date End Date Vehicle Control HFD PBS Week-9 Week-20(N = 10) REMD2.59C HFD REMD2.59C, Week-9 Week-20 (N = 10) 7.5 mg/kg PairFeeding HFD PBS Week-9 Week-20 (N = 10) Prevention HFD REMD2.59C, Week-1Week-20 (N = 8) 7.5 mg/kg Normal Diet Normal Diet PBS Week-9 Week-20 (N= 10)

Body weight is measured weekly throughout the study. Food consumption(food in/food out) is recorded weekly throughout the study. Foodconsumption is monitored daily for the first week post treatment for the“REMD2.59 Group” and the amount on post-treatment day 7 is then used tofeed the “Pair Feeding Group” during week 10. Each week thereafter, foodconsumption of the “REMD2.59 Group” is monitored weekly and the foodconsumption amount at the end of each week is used to feed the “PairFeeding Group” the following week. The chow fed “Normal diet group” isfed the same amount of chow throughout the entire 20 week study.

In addition to body weight and food consumption, various otherparameters are measured throughout the 20 week study, including, e.g.,i) fasting blood glucose determination was measured via tail veinsweekly using Accu-Chek Aviva System® (mice are fasted for 6 hours priorto the test and fasting blood glucose levels); ii) oral glucosetolerance test (OGTT) was performed for all animals at the end of thestudy to test the repeat dosing effect of REMD Ab2.59. The baseline(time 0) glucose level was measured after 16 hours fast and prior toglucose challenge. Following oral administration of 2 g/kg glucose, theblood glucose levels were measured at different time points (30, 60, 120min) by using Accu-Chek Performa System; iii) the lipid profile in serumand blood bio-chemistry parameters (ALT, AST, GGT, ALP, TG and TCHO)were tested throughout the study. Blood samples were obtained on week 8,12, 16 and 20, the samples were immediately processed by centrifugationat 4° C., 4000 g for 15 minutes, and then they were transferred into newtest tubes. Lipid profile and blood bio-chemistry parameters weremeasured by using TOSHIBA TBA-40FR automated biochemical analyzer; iv)the lipid profiles (TG, TCHO, HDL-C and LDL-C) were extracted from theliver of the animal according to the protocol, and then lipid profilewere measured by using TOSHIBA TBA-40FR automated biochemical analyzer;v) the insulin level of all study animals were measured on week 8, 12,16 and 20. GLP-1 and leptin were measured at the end of the study withELISA method. The blood serum was used for the analysis; and vi) on thetermination day, after OGTT study necropsy was conducted. At the end ofthe study, tissue or organs were collected and the wet weights of thepancreas, white adipose tissue (WAT), muscle (gastrocnemius muscle) andliver were measured. Half of these tissue samples were fixed and broughtup to paraffin block for H&E (liver and WAT) or IHC (pancreas) analysis.Hypothalamus, brain, heart and remaining part of pancreas, WAT, muscle,liver were stored at −80° C. or future analysis. The variousmeasurements and/or analysis described above is made as described in theAdditional Materials and Methods section below. All statistical testswere conducted, and the level of significance were set at 5% or P<0.05.The group means and standard deviation were calculated for allmeasurement parameters as study designed. A one-way analysis of variance(ANOVA) was used among the groups with software Graph Pad Prism 5.0.

Results

Body Weight and Food Consumption—

As depicted in FIG. 1, the REMD2.59 Group efficiently reduced weightgain from Week 9 to Week 20. The Pair Feeding Group, which was given thesame amount of daily food as that of the REMD2.59 Group from Week 9 toWeek 20, showed a similar, but slightly greater weight gain, than theREMD2.59 Group. This observation suggests that the effects of REMD2.59are not limited to the reduction in food intake. The Prevention Group,which received REMD2.59 weekly injections concurrently with HFD fromWeek 1 through Week 20, showed the lowest weight gain compared with allother groups, despite the HFD feeding. Specifically, the average bodyweight of the Prevention Group (35.3±3.0 g) was 34% lower (P<0.01) thanthe Vehicle Group (53.3±2.4 g), and 9% lower even than the Normal DietGroup (38.6±3.1 g), at the end of the study (on Day 140, or end ofWeek-20). As depicted in FIG. 2, the Prevention Group consumed the sameamount of calories per gram of body weight as the Normal Diet Group. Onthe other hand, the HFD fed groups (Vehicle Group, REMD2.59 Group andPair Feeding Group) all consumed nearly the same amount of calories whenadjusted by gram of body weight. Nevertheless, taking into considerationof the body weight differences, the Vehicle Group still consumes greateramount of calories per animal, than the REMD2.59 Group and Pair FeedingGroups, since the Vehicle Group has the highest average body weight.

Fasting Blood Glucose—

As depicted in FIG. 3, REMD2.59 treatment initiated from Week 9 (i.e.,the REMD2.59 Group) resulted in markedly lower blood glucose levels thanthe Vehicle Control Group. This correction of hyperglycemia cannot beexplained by the reduced energy consumption alone, since the PairFeeding Group achieved a much smaller magnitude of glucose lowering thanthe REMD2.59 Group. The Prevention Group led to the lowest fastingplasma glucose profiles, even lower than the Normal Diet Control Group.Finally, diet-induced obesity is clearly associated with hyperglycemia,as evidenced by the Vehicle Control Group.

Blood Glucose Levels from Oral Glucose Tolerance Test (OGTT)—

As depicted in FIG. 4, REMD 2.59, given either as treatment (REMD2.59Group) or as a preventive measure (Prevention Group), resulted inmarkedly lower blood glucose profiles during an oral glucose tolerancetest (OGTT). However, the untreated (Vehicle Group) or food-restricted(Pair-Fed Group) displayed a diabetic OGTT glucose profile, featuringelevated baseline glucose levels and higher glucose excursions duringthe OGTT, and ending in higher post-OGTT glucose profiles. As depictedin FIG. 5, the OGTT glucose area under the curve (AUC) values appear toconfirm the observations, and support the observations depicted in FIG.4.

Lipid Profile and Blood Biochemistry Data—

As depicted in FIG. 6, triglyceride (TG) levels were not significantlydifferent between the HFD-fed Vehicle Group and the Normal Diet ControlGroup. REMD2.59 Treatment did not significantly affect TG levels, andalthough the Prevention Group showed reduced TG levels at Week 16, thechanges were not sustained by the end of study. Thus, overall REMD2.59did not cause any major change in circulating TG levels. As depicted inFIG. 7, the total cholesterol (TCHO) levels were not affected byREMD2.59 Treatment (comparing Vehicle Group vs. REMD2.59 Group), but wassignificantly reduced by the Prevention Group (up to 47% by Week 20) toa level close to that of the Normal Diet Group.

Lipid Profile in Liver Analysis—

As depicted in FIG. 9, REMD2.59 treatment moderately, althoughstatistically insignificantly, reduced TG content in the liver tissue(102.0±45.2 vs. 131.6±46.5 mg/g tissue) in comparison to the VehicleGroup mice. The Prevention Group showed a reduced the liver TG contentby 84% (21.4±14.5 vs. 131.6±46.5 mg/g tissue) in comparison to theVehicle Group. As depicted in FIG. 10, the total cholesterol (TCHO),high density and low density lipoprotein levels (HDL-C and LDL-C) werenot significantly affected by REMD2.59 Treatment or Prevention.

Measurement of GLP-1, Insulin and Leptin—

As depicted in FIG. 11, throughout the treatment period and up until theend of the 20-week study, both the REMD2.59 Group and the PreventionGroup demonstrated a very robust effect in correcting hyperinsulinemia,as insulin levels were brought back to, or even lower than, the levelsseen in the Normal Diet Group. This demonstrates an important biologicalaction for REMD2.59, as in human obesity, type 2 diabetes, andNAFLD/NASH, dyslipidemia is closely associated with hyperinsulinemia. Asdepicted in FIG. 12, leptin were reduced in the Prevention Groupcompared to the Vehicle Group, by as much as 92%, and the reduced leptinlevel was even lower than that of the Normal Diet Group. This issignificant in that leptin levels in circulation signifies the extent ofadiposity. The REMD2.59 Group reduced leptin levels slightly, althoughstatistically insignificantly. As depicted in FIG. 13, the REMD2.59Group, but not the Prevention Group, is associated with a 10-foldincrease in the circulating active GLP-1 levels, suggesting acontribution by GLP-1 in controlling food intake and other metabolicbenefits by blockade of glucagon receptors using REMD2.59. Indeed,REMD2.59 weekly treatment induced a nearly 10-fold increase in thecirculating active GLP-1 level, which may account for the additionalweight reduction effects.

Histology and Immunohistochemistry Results—

As depicted in FIG. 14, the Prevention Group exhibited a significantlyreduced the white adipose tissue weight, suggesting that blockade ofglucagon receptor is associated with lower adiposity. The PreventionGroup also exhibited reduced liver wet weight, which may be related tothe reduced fat (TG) and glycogen contents. The REMD2.59 Group exhibitedreduced wet liver weight, similarly to the Prevention Group, butslightly increased the pancreatic wet tissue weight, which might be dueto the reactive increase in the islet tissue mass. As depicted in FIG.15, in the immunochemistry (IHC) stained pancreatic sections, the REMD2.59 Group exhibited reduced insulin area/islet area, similar to thePrevention Group, collectively suggesting that blockade of glucagonreceptor signaling attenuated the stimulation to islet beta-cells and toinsulin synthesis/secretion. Such a histological finding is in line withthe observation that the REMD2.59 Group and Prevention Group exhibitedlower insulin levels in circulation (FIG. 11, above). On the other hand,both the REMD2.59 Group and Prevention Group induced marked increases inglucagon area/islet area, suggesting a reactive feedback stimulation ofglucagon synthesis/secretion from the islet alpha-cells.

As depicted in FIG. 16, the following observations can be derived: 1)the liver tissues from the Vehicle Group show abundance of vacuoles dueto fat droplets, which confirms the fatty liver diagnosis of these mice;2) the liver samples from the REMD2.59 Group appear to show markedlyreduced density and sizes of fat droplets, i.e., the areas occupied byovert fat droplets appear to be much smaller than those of the VehicleGroup; 3) the liver tissue samples from the Pair Feeding Group show verylittle, if any, reduction in the density and sizes of fat droplets fromthose observed in the Vehicle Group; 4) the liver tissue samples fromthe Normal Diet Group show very clean liver sections, with virtually novisible fat droplets, comparable to those of the Prevention Group; and5) the liver tissue samples from the Prevention Group show very cleanliver sections, with virtually no visible fat droplets, comparable tothose of the Normal Diet Group. The above histological evidence clearlyindicates the robust effects of the antagonistic glucagon receptorantibody in correcting, or preventing, the fatty liver changes in thediet-induced obese mice. The histological improvement in fatty liver, asevidenced by the liver histology, is closely associated with thecorrection of hyperglycemia and hyperinsulinemia, and improvements inglucose and fat metabolism.

Example 2

In view of the significant effects of REMD2.59C treatment demonstratedin Example 1, the relationship between regulating glucose output and thedevelopment of NAFLD/NASH in various murine models is evaluated. In thisexample, the in vivo activity of REMD2.59C is evaluated in theNASH-derived HCC murine model (STAM™ model, Fujii et al, Med MolMorphol, 46:141-152, 2013). In STAM™ model, C57BL/6J mice are injectedwith a single subcutaneous injection of 200 μg STZ at 2 days after birthand put on a HFD or chow after 4 weeks of age. In male mice, thiscombined STZ-HFD treatment results in the development of steatosis anddiabetes after 1 week after feeding HFD, and with continued HFD the malemice develop fibrosis, cirrhosis and hepatocellular carcinoma (HCC)along with hyperglycemia and moderate hyperlipidemia, thus closelyresembling human NASH. The male mice treated only with STZ and thefemale mice treated with STZ-HFD develop diabetes, but not HCC.

In this study, four groups of 10 each wild-type C57BL/6J mice (male, age4-6 weeks, 20-22 g) are injected with a single subcutaneous injection of200 μg STZ at 2 days after birth and put on a HFD after 4 weeks of age(“STZ-HFD groups”) and one group of 10 wild-type C57BL/6J mice (male,age 4-6 weeks, 20-22 g) are injected with a single subcutaneousinjection of 200 μg STZ at 2 days after birth and fed with normal diet(chow) after 4 weeks of age (“STZ-Chow group”). The STZ-Chow groupcontinues on chow throughout the 24 week study and the STZ-HFD groupscontinue on HFD throughout the 24 week study.

At age 5 weeks, the STZ-Chow group is dosed weekly via subcutaneousinjection with vehicle (PBS) up until age 24 weeks, and one STZ-HFDgroup is dosed weekly with 7.5 mg/kg REMD2.59 antibody up to age 24weeks. At age 8 weeks, one STZ-HFD group is dosed weekly with 2.5 mg/kgREMD2.59 antibody up to age 24 weeks, one STZ-HFD group is dosed weeklywith 5.0 mg/kg REMD2.59 antibody up to age 24 weeks, and one STZ-HFDgroup is dosed weekly with 7.5 mg/kg REMD2.59 antibody up to age 24weeks.

Various parameters are measured throughout the 24 week study, including,e.g., i) body weight (once a week); ii) fasting blood glucosedetermination (mice are fasted for 6 hours prior to the test and fastingblood glucose levels are measured via tail veins weekly using Accu-ChekAviva System®; iii) serum hemoglobin-A1c (HbA1c) determination; iv)serum GLP-1 determination; v) serum insulin and leptin levels viaradioimmunoassay (Linco, St. Charles, Mo.); vi) serum alanineaminotransferase (ALT) determination; vii) serum adioponectindetermination; viii) serum lipids (e.g., total cholesterol, LDL, HDL andtriglycerides) determination; and ix) gamma-glutamyl transpeptidase(CGT) determination. For items iii)-ix), blood samples are collectedpre-dose and at the end of the study into tubes without anyanticoagulant, immediately centrifuged and the serum transferred intoseparate sample tubes for evaluation. The various measurements and/oranalysis described above is made as described in the AdditionalMaterials and Methods section below.

At the end of the study, livers are rapidly excised, rinsed in ice-coldsaline, and weighed. Aliquots of liver are snap frozen in liquidnitrogen and kept at −80° C. until being analyzed. A portion of eachliver is fixed in 10% formalin for proper histological analysis of theliver. Liver triglyceride (TG) content, diacylglyceride (DG) content,and ceramide content measurements are made as described in theAdditional Materials and Methods section below. Inflammation, centralvein fibrosis, and portal tract fibrosis will be evaluated as describedin the Additional Materials and Methods section below.

In view of the results demonstrated in Example 1, it is expected thattreatment of the wild-type mice using an anti-GCGR antibody will providebeneficial therapeutics effects which may include, e.g., reducinginsulin resistance; reducing or preventing hyperinsulinemia, reducing orpreventing fat deposits in the liver; reducing or preventinginflammation in the liver; reducing or preventing the accumulation oflipid, e.g., hepatic triacylglycerol, hepatic diacylglycerol, andceramides; and preventing injury in the liver, and that the developmentof NAFLD/NASH in such mice may be prevented or treated, thus reducingthe risk of the diabetic subject from developing HCC.

Example 3

In view of the significant effects of REMD2.59C treatment demonstratedin Example 1, the in vivo activity of REMD2.59C is evaluated in a murinemodel of NASH, using db/db mice. The study will be a 24 week study. Anadditional 48 week study may also be performed. db/db mice from theC57BL/6 background are purchased from Jackson Laboratory (Bar Harbor,Me.). db/db mice are hyperleptinemic, obese and diabetic mice. db/dbmice that are fed a methionine and choline deficient (MCD) dietspontaneously develop hepatic steatosis, which progresses to NASH(Wortham et al., Dig Dis Sci., 53(10): 2761-2774, 2008 October). Sixmice each of db/db at age 10-12 weeks are fed ad libitum with either amethionine and choline deficient (MCD) diet (MP Biomedicals Solon, Ohio,cat. no. 960439) or the same diet supplemented with methionine andcholine (MCDS) diet (MP Biomedicals cat. no. 960441) for 4 weeks, or aHFD+fructose Western Diet (WD)(#58Y1, TestDiet, St Louis, Mo.), or chow(Control Diet)(#58Y2, TestDiet, St Louis, Mo.) for 24 weeks. Mice arehoused individually in steel microisolator cages at 22° C. with a12-h/12-h, light/dark cycle. All procedures were conducted in accordancewith the National Institutes of Health (NIH) guidelines for the care anduse of laboratory animals. The mice are dosed weekly or bi-weekly viasubcutaneous injection with either vehicle (10 mM sodium acetate, 5%sorbitol, and 0.004% polysorbate 20), 2.5 mg/kg REMD2.59C antibody (“LowDose”), or 5 mg/kg REMD2.59C antibody (“High Dose”) for 4 weeks or 24weeks, as appropriate. The dose administered will not exceed 10 mg/kgper month.

Various parameters are measured throughout the 4 week or 24 week study,including, e.g., i) body weight (once a week); ii) fasting blood glucosedetermination (mice are fasted for 6 hours prior to the test and fastingblood glucose levels are measured via tail veins weekly using Accu-ChekAviva System®; iii) serum hemoglobin-A1c (HbA1c) determination; iv)serum GLP-1 determination; v) serum insulin and leptin levels viaradioimmunoassay (Linco, St. Charles, Mo.); vi) serum alanineaminotransferase (ALT) determination; vii) serum adioponectindetermination; viii) serum lipids (e.g., total cholesterol, LDL, HDL andtriglycerides (TG)) determination; and ix) gamma-glutamyl transpeptidase(CGT) determination. For items iii)-ix), blood samples are collectedpre-dose and at the end of the study into tubes without anyanticoagulant, immediately centrifuged and the serum transferred intoseparate sample tubes for evaluation. The various measurements and/oranalysis described above is made as described in the AdditionalMaterials and Methods section below.

At the end of the study, livers are rapidly excised, rinsed in ice-coldsaline, and weighed. Aliquots of liver are snap frozen in liquidnitrogen and kept at −80° C. until being analyzed. A portion of eachliver is fixed in 10% formalin for proper histological analysis of theliver. Liver triglyceride (TG) content, diacylglyceride (DG) content,and ceramide content measurements are made as described in theAdditional Materials and Methods section below. Inflammation, centralvein fibrosis, and portal tract fibrosis will be evaluated as describedin the Additional Materials and Methods section below.

In view of the results demonstrated in Example 1, it is expected thattreatment of the db/db mice using an anti-GCGR antibody will providebeneficial therapeutics effects which may include, e.g., reducinginsulin resistance; reducing or preventing hyperinsulinemia, reducing orpreventing fat deposits in the liver; reducing or preventinginflammation in the liver; reducing or preventing the accumulation oflipid, e.g., hepatic triacylglycerol, hepatic diacylglycerol, andceramides; and preventing injury in the liver, and that the developmentof NAFLD/NASH in such mice may be prevented or treated.

Example 4

This Example describes a randomized, double-blind, placebo-controlled,parallel group, multiple dose study to evaluate the safety,pharmacokinetics and pharmacodynamic effects of weekly treatment using afully human anti-GCGR antibody in subjects diagnosed with NASH. Thetreatment may last a period up to 6 or 12 months, long enough to observeand quantitate treatment efficacy and safety.

Treatment groups include a placebo group and treatment groups to betreated with various dosages of a fully human anti-GCGR antibody whichcomprises the heavy chain sequence set forth in SEQ ID NO: 51 and thelight chain sequence set forth in SEQ ID NO: 52 (“REMD-477”). Examplesof non-placebo treatment groups will include, e.g., subjects who receiveinjections of either 0.01 mg/kg, 0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg,0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, or 10 mg/kg REMD-477 per week, andsubjects who receive injections of either 0.01 mg/kg, 0.025 mg/kg, 0.05mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1.0mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, or 10 mg/kgREMD-477 weekly.

Primary outcome measures will include, e.g., change in percentage ofliver fat content by MRI at: baseline, at 4-week or 8-week intervals,until the end of study; change in the proportion of REMD-477 treatedpatients relative to placebo achieving improvement of liver fibrosis byat least one stage, at the end of study, in comparison to the baselineassessment; and change in liver enzyme and metabolic markers, includingAspartate Transaminase (AST), Alanine Transaminase (ALT), Bilirubin andAlkaline phosphatase (ALP), at: baseline, at 4-week intervals, until theend of study. Secondary outcome measures will include, e.g., change infasting plasma glucose levels, average of daily morning glucose after anovernight fast, at pre-treatment baseline, and at weekly intervals untilthe end of study; change in plasma insulin levels at pre-treatmentbaseline, and at weekly intervals until the end of study; change inhemoglobin A1c levels (indicator of chronic glucose control) atpre-treatment baseline, and at 4-week or 8-week intervals until the endof study; change in glucose profiles at oral glucose tolerance tests(OGTT), measured at 0, 30, 60, 90 and 120 min after an oral glucoseload, at pre-treatment baseline, and at 8-week intervals until the endof study; composite long term outcome measured by the number of patientswith the onset of any adjudicated events, including cirrhosis, all-causemortality, and liver-related clinical outcomes, at the Baseline and theend of study; and changes in scores of the Quality of Life (36-ItemShort-Form Health Survey [SF-36]) Questionnaire.

Additional Materials and Methods

Body Weights:

Body weights of all animals are measured weekly throughout the durationof the various studies.

Food Consumption:

Food consumption of all animals are measured daily and/or weeklythroughout the duration of the various studies.

Blood Glucose:

The mice were fasted 6 hours prior to blood glucose test from 9 am to 3pm, and fast blood glucose levels were measured via tail veins on weeklybasis by using Accu-Chek Performa System.

Oral Glucose Tolerance Test:

To test the repeat dosing effect of REMD Ab2.59, OGTT was performed forall animals at the end of the study. The baseline (time 0) glucose levelwas measured after 16 hours fast and prior to glucose challenge.Following oral administration of 2 g/kg glucose, the blood glucoselevels were measured at different time points (30, 60, 120 min) by usingAccu-Chek Performa System.

Blood Chemistry Analysis:

The lipid profile of every 2^(nd) week and terminal blood bio-chemistryparameters (ALT, AST, GGT, ALP) were tested. Blood samples were obtainedon week 8, 12, 16 and 20, the samples were immediately processed bycentrifugation at 4° C., 4000 g for 15 minutes, and then they weretransferred into new test tubes. Lipid profile and blood bio-chemistryparameters were measured by using TOSHIBA TBA-40FR automated biochemicalanalyzer.

Measurement of Lipid Profile in Liver:

The lipid profiles (TG, TCHO, HDL-C and LDL-C) were extracted from theliver of the animal according to the protocol, and then lipid profilewere measured by using TOSHIBA TBA-40FR automated biochemical analyzer.

ELISA Kits Analysis:

The insulin level of all study animals were measured on week 8, 12, 16and 20. GLP-1 and leptin were measured at the end of the study withELISA method. The blood serum was used for the analysis.

Liver Weight:

At the end of the study, livers are rapidly excised, rinsed in ice-coldsaline, and weighed. Aliquots of liver are snap frozen in liquidnitrogen and kept at −80° C. until being analyzed. A portion of eachliver is fixed in 10% formalin for histology.

Liver TG/DG/Ceramide Content:

Liver triglyceride (TG), diacylglyceride (DG), and ceramide content ofall animals are measured at the end of the study. Liver samples arehomogenized in 50 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl, 1mM EDTA, and 1 μM PMSF and lipids isolated by extraction into chloroformwith appropriate internal standards included for each protocol.Extracted lipids were resuspended and diluted in methanol/chloroform(4:1, by volume) before analysis by electrospray ionization-massspectrometry using a Thermo Electron TSQ Quantrum Ultra instrument (SanJose, Calif.). DG molecular species were quantified as sodiated adductsusing selected reaction monitoring as previously described withintensity of each species normalized to that of the internal standarddi-20:0 DG (Demarco V G et al., Endocrinology, 154:159-171, 2013). TGaliphatic groups were quantified by TG fingerprinting techniques withneutral loss scanning for the loss of each fatty acid from the TGspecies and comparisons to that of the neutral loss 268 which is derivedfrom the internal standard Tri-17:1 TG (Han et al., Anal Biochem,295:88-100, 2001). Individual ceramide molecular species were quantifiedin negative ion mode using neutral loss 256 by comparing the ionintensity of individual molecular species to that of the internalstandard (17:0 ceramide) after corrections for type I and type II ¹³Cisotope effects.

Histology and Immunohistochemistry:

Formalin-fixed liver tissue is processed, and 5-μm-thick paraffinsections are stained with hematoxylin and eosin (H&E) and Masson'strichrome for histological analysis. Inflammation is evaluated onH&E-stained sections and is given a score from 0 to 3 as follows: 0, noinflammation; 1, mild; 2, moderate; 3, severe. The degree of fibrosis isassessed by digital morphometry. Five discrete regions of atrichrome-stained sections from each mouse are randomly selected andwithin each region identified a portal track and central vein to bedigitally photographed. Photographs are obtained with a ×20 objectivewith the portal tracks or central veins of interest in the center of thefield, thus obtaining five portal/periportal and fivecentral/pericentral fields of interest for each mouse. For each field ofinterest the pixels corresponding to fibrosis are measured based on anarrow band of the blue spectrum corresponding to the stain of clear-cutfibrosis in each specimen, carefully excluding the normal stromalcollagen in those areas. The number of pixels corresponding to fibrosisis measured as a percentage of the total pixels of each image using theImage Processing Tool Kit, version 5.0 (Reindeer Graphics, Asheville,N.C.). The results of the five portal/periportal and fivecentral/pericentral fields from each specimen are averaged, and fibrosisis expressed as a percentage of total cross-sectional area for eachanimal.

Methionine and Choline Deficient (MCD) Diet:

Of the dietary approaches discussed herein, MCD diets produce the mostsevere NASH phenotype in the shortest time frame. MCD diet is high insucrose and fat, but lacks methionine and choline, which are essentialfor hepatic beta-oxidation and the production of very low densitylipoprotein (VLDL). This results in the accumulation of intra-hepaticlipid and decreased VLDL synthesis (Anstee et al., Int J Exp Pathol,87(1):1-16, 2006). MCD diets will quickly induce measurable hepaticsteatosis (mainly macrovesicular) in rodents by 2-4 weeks and thisprogresses to inflammation and fibrosis shortly thereafter. Fat levelsin MCD diets can vary, though typically they contain about 20% fat byenergy. Importantly, unlike human or other diet-induced rodent models ofNAFLD, rodents fed MCD diets lose weight (due to a vastly lower caloricintake) and do not become insulin resistant. Since most humans with NASHare obese and insulin resistant, this represents an important differencein how MCD diets model human NASH.

High-Fat Diets (HFD):

HFD are well-known to increase body weight, body fat and induce insulinresistance in rodent models. HFD can also increase liver fat levelsquite rapidly (within days) as well as hepatic insulin resistance beforesignificant increases in peripheral fat deposition occur. Chronically,HFD-induced liver fat accumulation may not follow a linear progressionand liver fat levels may actually decrease, then increase again duringprolonged HFD feeding. When fed for equal lengths of time, HFD feedingresults in 10-fold lower liver fat levels compared to what accumulateson an MCD diet. In general, HFD feeding does not produce liver fibrosisand only mild steatosis as compared to MCD diets, thus highlighting animportant difference between these dietary regimes. It is important toremember that the term ‘HFD’ encompasses a wide variety of diet formulasand diets of different composition can be expected to alter the liverphenotype in various ways. An exemplary HFD diet may consist of 36% fatderived-calories (9% corn oil and 27% butter) and 43.2%carbohydrate-derived calories without sugar. HFD (Research Diets,D12492, HFD) was used in these studies.

HFD+Fructose Diet (Western Diet (“WD”)):

An exemplary WD may consist of 36% fat derived-calories (9% corn oil and27% butter), which is the same as HFD, and 43.2% carbohydrate-derivedcalories with e.g., fructose (e.g., 30% sugar-derived calories).

Murine Models of NASH:

The anti-GCGR antibodies of the present disclosure may be evaluated inany of the other various published murine models of NASH (see, e.g.Poekes et al., Archives of Public Health, 72(1): 07, 2014; Adorini etal., Drug Discovery Today, 17:988-997, 2012; Farrell et al., Liver Int.,34(7):1084-93, 2014; Rooyen et al, Gastroenterology, 141(4):1393-1403,2011; Ishimoto et al., Hepatology, 58(5):1632-1643, 2013; Farrell etal., Gut and Liver, 6(2):149-171, 2012; Sahai et al., Am J PhysiolGastrointest Liver Physiol, 287:G1035-G1043, 2004; Wortham et al., DigDis Sci, 53(10):2761-2774, 2008; Lieber et al, Am J Clin Nutr,79:502-509, 2004).

All of the articles and methods disclosed and claimed herein can be madeand executed without undue experimentation in light of the presentdisclosure. While the articles and methods of this disclosure have beendescribed in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to the articlesand methods without departing from the spirit and scope of thedisclosure. All such variations and equivalents apparent to thoseskilled in the art, whether now existing or later developed, are deemedto be within the spirit and scope of the disclosure as defined by theappended claims. All patents, patent applications, and publicationsmentioned in the specification are indicative of the levels of those ofordinary skill in the art to which the disclosure pertains. All patents,patent applications, and publications are herein incorporated byreference in their entirety for all purposes and to the same extent asif each individual publication was specifically and individuallyindicated to be incorporated by reference in its entirety for any andall purposes. The disclosure illustratively described herein suitablymay be practiced in the absence of any element(s) not specificallydisclosed herein. Thus, for example, in each instance herein any of theterms “comprising”, “consisting essentially of”, and “consisting of” maybe replaced with either of the other two terms. The terms andexpressions which have been employed are used as terms of descriptionand not of limitation, and there is no intention that in the use of suchterms and expressions of excluding any equivalents of the features shownand described or portions thereof, but it is recognized that variousmodifications are possible within the scope of the disclosure claimed.Thus, it should be understood that although the present disclosure hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis disclosure as defined by the appended claims.

SEQUENCE LISTINGS

The amino acid sequences listed in the accompanying sequence listing areshown using standard three letter code for amino acids, as defined in 37C.F.R. 1.822. This disclosure includes a Sequence Listing in computerreadable form (ST25 format text file) prepared through the use ofsoftware program PatentIn and is identical to the accompanying sequencelistings.

SEQ ID NO: 1 is the amino acid sequence of a human glucagon receptor(GCGR) molecule (Accession Number AA104855).

SEQ ID NO: 2 is the amino acid sequence encoding the heavy chainvariable region of a fully human anti-GCGR antibody. SEQ ID NO: 3 is theamino acid sequence encoding the light chain variable region of a fullyhuman anti-GCGR antibody.

SEQ ID NO: 4 is the amino acid sequence encoding the heavy chainvariable region of a fully human anti-GCGR antibody. SEQ ID NO: 5 is theamino acid sequence encoding the light chain variable region of a fullyhuman anti-GCGR antibody.

SEQ ID NO: 6 is the amino acid sequence encoding the heavy chainvariable region of a fully human anti-GCGR antibody. SEQ ID NO: 7 is theamino acid sequence encoding the light chain variable region of a fullyhuman anti-GCGR antibody.

SEQ ID NO: 8 is the amino acid sequence encoding the heavy chain of achimeric anti-GCGR antibody. SEQ ID NO: 9 is the amino acid sequenceencoding the light chain of a chimeric anti-GCGR antibody.

SEQ ID NOS: 10-28 are amino acid sequences encoding the heavy chainvariable regions of various fully human anti-GCGR antibodies.

SEQ ID NOS: 29-47 are amino acid sequences encoding the light chainvariable regions of various fully human anti-GCGR antibodies.

SEQ ID NO: 48 is the amino sequence encoding the kappa light chainconstant region. SEQ ID NO: 49 is the amino sequence encoding the lambdalight chain constant region.

SEQ ID NO: 50 is the amino sequence encoding the IgG2 heavy chainconstant region.

SEQ ID NO: 51 is the amino acid sequence encoding the heavy chain of ahuman anti-GCGR antibody. SEQ ID NO: 52 is the amino acid sequenceencoding the light chain of a human anti-GCGR antibody.

SEQUENCE LISTINGS SEQ ID NO: 1-Amino acid sequence of a human glucagon receptor (GCGR) moleculeMPPCQPQRPLLLLLLLLACQPQVPSAQVMDFLFEKWKLYGDQCHHNLSLLPPPTELVCNRTFDKYSCWPDTPANTTANISCPWYLPWHHKVQHRFVFKRCGPDGQWVRGPRGQPWRDASQCQMDGEEIEVQKEVAKMYSSFQVMYTVGYSLSLGALLLALAILGGLSKLHCTRNAIHANLFASFVLKASSVLVIDGLLRTRYSQKIGDDLSVSTWLSDGAVAGCRVAAVFMQYGIVANYCWLLVEGLYLHNLLGLATLPERSFFSLYLGIGWGAPMLFVVPWAVVKCLFENVQCWTSNDNMGFWWILRFPVFLAILINFFIFVRIVQLLVAKLRARQMHHTDYKFRLAKSTLTLIPLLGVHEVVFAFVTDEHAQGTLRSAKLFFDLFLSSFQGLLVAVLYCFLNKEVQSELRRRWHRWRLGKVLWEERNTSNHRASSSPGHGPPSKELQFGRGGGSQDSSAETPLAGGLPRLAESPFSEQ ID NO: 2-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTTVTVSSSEQ ID NO: 3-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIKSEQ ID NO: 4-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTTVTVSSSEQ ID NO: 5-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFVTYYCLQHNSNPLTFGGGTKVEIKSEQ ID NO: 6-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTTVTVSSSEQ ID NO: 7-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLESGVPSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIKSEQ ID NO: 8-Amino acid sequence of a heavy chain of a chimeric antibody that binds GCGRMEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKSEQ ID NO: 9-Amino acid sequence of a light chain of a chimeric antibody that binds GCGRMDMRVPAQLLGLLLLWFPGARCDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLESGVPSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECSEQ ID NO: 10-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVILSDGRNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDDYEILTGYGYYGMDVWGQGTTVTV SSSEQ ID NO: 11-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVILNDGRNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDDYEILTGYGYYGMDVWGQGTTVTV SSSEQ ID NO: 12-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNGAAWNWIRQSPSRGLEWLGRTYYRSKWYYDYAGSVKSRININPDTSKNQFSLQVNSVTPEDTAVYYCTRDRSSGWNEGYYYYGMDVWGQG TTVTVSSSEQ ID NO: 13-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDIHWVRQAPGKGLEWVAVLSSDGNNKYCADSVKGRFTISRDNSKNTLYLQMNSLRTEDTAVYYCAREEVYYDILTGYYDYYGMDVWGQGTTV TVSSSEQ ID NO: 14-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLQESGPGLVKPSETLSLTCTVSGGSISTYFWTWIRQFPGKGLEWIGYIFYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGYYDILTGEDYSYGMDVWGQGTTVTVSSSEQ ID NO: 15-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLQQSGPGLVKPSQILSLICAISGDRVSSNGAAWNWIRQSPSRGLEWLGRTYYRSKWYYDYAGSVKSRININPDTSKNQFSLQVNSVTPEDTAVYYCARDRSSGWNEGYYYYGMDVWGQGT TVTVSSSEQ ID NO: 16-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLQESGPGLVKPSETLSLTCTVSGGSISTYFWTWIRQFPGEGLEWIGYIFYSGNTNYNPSLTSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGYYDILTGEDYSYGIDVWGQGTTVTVSSSEQ ID NO: 17-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFIFSSYGMHWVRQAPGKGLEWVAVISNDGSNKYYADFVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDYDILTGNGVYGMDVWGQGTTVTV SSSEQ ID NO: 18-Amino acid sequence of a HCVR of a human antibody that binds GCGREVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMNWVRQAPGKGLEWVSYISGSSSLIYYADSVKGRFTISRDNAKNSLYLHMNSLRDEDTAVYYCARARYNWNDYYGMDVWGQGTTVTVSSSEQ ID NO: 19-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGIHWVRQAPGKGLEWVAGIWYDGSNKYYADSVKGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCARLFDAFDIWGQGTMVTVSSSEQ ID NO: 20-Amino acid sequence of a HCVR of a human antibody that binds GCGREVQLVESGGGLVQPGGSLRLSCAASGFIFSSYTMNWVRQAPGKGLEWVSYISSSSSLIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARSDYYGSGSYYKGNYYGMDVWGQGTTV TVSSSEQ ID NO: 21-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVTIIWSDGINKYYADSVKGRFTISRDNSKNTLNLQMNSLRAEDTAVYYCARERGLYDILTGYYDYYGIDVWGQGTTVT VSSSEQ ID NO: 22-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVTIIWSDGINKYYADSVKGRFTISRDNSKNTLNLQMNSLRAEDTAVYYCARERGLYDILTGYYDYYGIDVWGQGTTVT VSSSEQ ID NO: 23-Amino acid sequence of a HCVR of a human antibody that binds GCGREVQLVESGGGLVKPGGSLRLSCAASGITFRSYSMNWVRQAPGKGLEWVSAISSSSSYIYYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCARGRYGMDVWGQGTTVTVSSSEQ ID NO: 24-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGSTFRSYDMHWVRQAPGKGLEWVAVISYDGSNKYYGDSVKGRLTISRDNSKNTLYLQMNSLRAEDTAVYYCARDQYDILTGYSSDAFDIWGQGTMVTV SSSEQ ID NO: 25-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGSHKYYEDSVKGRFTISRDNSKNTLYLQMNSLRADDTGVYYCARVGYGSGWYEYYYHYGMDVWGQGT TVTVSSSEQ ID NO: 26-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTT VTVSSSEQ ID NO: 27-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTT VTVSSSEQ ID NO: 28-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGITFSSYGMHWVRQAPGKGLEWVASIWYDGSNKYYVDSVKGRFTIFRDNSKKTLYLQMNRLRAEDTAVYYCARLGGGFDYWGQGTLVTVSSSEQ ID NO: 29-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWFQKKPGKAPKSLIYVVSSLQSGVPSRFSGSGSGTDFTLTINNLQPEDFATYYCQQYNHYPLTFGGGTRVEIKRSEQ ID NO: 30-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWFQQRPGKAPKSLIYVVSSLQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYFCQQYNHYPLTFGGGTKVEIKRSEQ ID NO: 31-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQFPSSLSASIGDRVTITCQASQDISNFLNWFQQKPGKAPKLLIYDASDLETGVPSRFSGSGAGTDFTFTISSLQPEDIATYFCQQYDDLPLTFGGGTRVDIKRSEQ ID NO: 32-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSNPLTFGGGTKVEIKRSEQ ID NO: 33-Amino acid sequence of a LCVR of a human antibody that binds GCGRQNVLTQSPGTLSLSPGERVTLSCRASQSVSSSYLAWYQQKPGQAPRLLIFGVSSRATGIPDRFSGSGSGTDFSLTISRLEPEDFAVYYCQQYGNSPFTFGPGTKVDIKRSEQ ID NO: 34-Amino acid sequence of a LCVR of a human antibody that binds GCGR DIQMTQFPSSLSASIGDRVTITCQASQDISNFLNWFQQKPGKAPKLLIYDASDLETGVPSRFSGSGAGTDFTFTISSLQPEDVATYFCQQYDNLPLTFGGGTKVDIKRSEQ ID NO: 35-Amino acid sequence of a LCVR of a human antibody that binds GCGRENVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLIFGVSSRATGIPDRFSGSGSGTDFSLTISRLEPEDFAVYYCQQYGNSPFTFGPGTKVDIKRSEQ ID NO: 36-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIDMYLAWFQQKPGKAPKSLIYAASSLQSGVPSKFSGSGFGTDFTLTISSLQPEDFATYYCQQYNIFPFTFGPGTKVDVKRSEQ ID NO: 37-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLESGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIKRSEQ ID NO: 38-Amino acid sequence of a LCVR of a human antibody that binds GCGRKIVMTQTPLALPVIPGEPASISCRSSQSLVDSDDGDTYLDWYLQKPGQSPQVLIHRLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMHRIEFPFTFGGGTKVEIKRSEQ ID NO: 39-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQRPGKAPKRLIYAASSLQTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIKRSEQ ID NO: 40-Amino acid sequence of a LCVR of a human antibody that binds GCGRGIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMEALQTMCSFGQGTKLEIKRSEQ ID NO: 41-Amino acid sequence of a LCVR of a human antibody that binds GCGR GIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMEALQTMSSFGQGTKLEIKRSEQ ID NO: 42-Amino acid sequence of a LCVR of a human antibody that binds GCGR DIVMTQTPLFLPVTPGEPASISCRSSQTLLDSDDGNTYLDWYLQKPGQSPQRLIYTLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQHIEFPSTFGQGTRLEIKRSEQ ID NO: 43-Amino acid sequence of a LCVR of a human antibody that binds GCGRSYELTQPPSVSVSPGQTASITCSGDKLGDKYASWYQQKPGQSPVLVIYQSTKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTVVFGGGTKLTVLGSEQ ID NO: 44-Amino acid sequence of a LCVR of a human antibody that binds GCGRNIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKNYLFWYLQKPGQSPQLLIYEVSYRFSGVPDRFSGSGSGTDFSLKISRVEAEDVGVYYCMQNIQPPLTFGQGTRLEIKRSEQ ID NO: 45-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIKRSEQ ID NO: 46-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLESGVPSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIKRSEQ ID NO: 47-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIVLTQTPLSLPVTPGEPASISCRSSQSLLDRDDGDTYLDWYLQKPGQSPQLLIYTLSYRASGVPDRFSGSGSGTDFSLKISRVEAEDVGVYYCMQRIEFPFTFGPGTKVDIKRSEQ ID NO: 48-Amino acid sequence of the constant light chain kappa regionRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 49-Amino acid sequence of the constant light chain lambda regionGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSSEQ ID NO: 50-Amino sequence of the IgG2 heavy chain constant regionASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 51-Amino acid sequence of a HC of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 52-Amino acid sequence of a LC of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVOLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

What is claimed is:
 1. A method for treating nonalcoholicsteatohepatitis (NASH) in a subject comprising administering to asubject diagnosed with NASH a therapeutically effective amount of anisolated fully human antibody that specifically binds to the humanglucagon receptor and which comprises the amino acid sequence encodingthe heavy chain of SEQ ID NO: 51 and the amino acid sequence encodingthe light chain of SEQ ID NO:
 52. 2. A method according to claim 1,wherein the therapeutically effective amount of the isolatedantagonistic antigen binding protein is selected from the groupconsisting of 0.001 to 100 mg/kg, 0.001 to 90 mg/kg, 0.001 to 80 mg/kg,0.001 to 70 mg/kg, 0.001 to 60 mg/kg, 0.001 to 50 mg/kg, 0.001 to 40mg/kg, 0.001 to 30 mg/kg, 0.001 to 20 mg/kg, 0.001 to 10 mg/kg, 0.001 to5 mg/kg, 0.001 to 4 mg/kg, 0.001 to 3 mg/kg, 0.001 to 2 mg/kg, and 0.001to 1 mg/kg body weight per week.
 3. A method according to claim 2,wherein the therapeutically effective amount of the isolatedantagonistic antigen binding protein is 0.01 to 10 mg/kg body weight perweek.
 4. A method according to claim 1, said method further comprisingadministering an anti-obesity agent to said subject, wherein theanti-obesity agent is selected from gut-selective microsomaltriglyceride transport protein (MTP) inhibitors, Cholecystokinin-A(CCKa) agonists, serotonin 5HT2c agonists, melanocortin 4 receptor(MCR4) agonists, lipase inhibitors, opioid antagonists, oleoyl-estrone,obinepitide, pramlintide (SYMLIN®), tesofensine, leptin, bromocriptine,orlistat, Advanced Obesity Drug-9604 (AOD-9604), and sibutramine.
 5. Amethod for treating nonalcoholic fatty liver disease (NAFLD) in asubject comprising administering to a subject diagnosed with NAFLD atherapeutically effective amount of an isolated fully human antibodythat specifically binds to the human glucagon receptor and whichcomprises the amino acid sequence encoding the heavy chain of SEQ ID NO:51 and the amino acid sequence encoding the light chain of SEQ ID NO:52.
 6. A method according to claim 5, said method further comprisingadministering an anti-obesity agent to said subject, wherein theanti-obesity agent is selected from gut-selective MTP inhibitors, CCKaagonists, serotonin 5HT2c agonists, MCR4 agonists, lipase inhibitors,opioid antagonists, oleoyl-estrone, obinepitide, pramlintide (SYMLIN®),tesofensine, leptin, bromocriptine, orlistat, AOD-9604, and sibutramine.7. A method of treating a subject classified as obese (e.g., having abody mass index (BMI) of 30 kg/m² or more) comprising administering tothe subject a therapeutically effective amount of an isolated fullyhuman antibody that specifically binds to the human glucagon receptorand which comprises the amino acid sequence encoding the heavy chain ofSEQ ID NO: 51 and the amino acid sequence encoding the light chain ofSEQ ID NO:
 52. 8. A method according to claim 7, said method furthercomprising administering an anti-obesity agent to said subject, whereinthe anti-obesity agent is selected from gut-selective MTP inhibitors,CCKa agonists, serotonin 5HT2c agonists, MCR4 agonists, lipaseinhibitors, opioid antagonists, oleoyl-estrone, obinepitide, pramlintide(SYMLIN®), tesofensine, leptin, bromocriptine, orlistat, AOD-9604, andsibutramine.